Digital Spatial Profiling Technology Research Articles

Potential Molecular Markers Related to Lymph Node Metastasis and Stalk Resection Margins in Pedunculated T1 Colorectal Cancers Using Digital Spatial Profiling: A Pilot Study with a Small Case Series.

There is a debate regarding the prediction of lymph node metastasis (LNM) in pedunculated T1 colorectal cancer (CRC). In this study with four cases of pedunculated T1 CRCs, we aimed to investigate gene expression variations based on the distance from the Haggitt line (HL) and identify potential molecular risk factors for LNM. By leveraging the Cancer Transcriptome Atlas and digital spatial profiling technology, we meticulously analyzed discrete regions, including the head, HL, proximal stalk region (300-1000 μm from HL), and distal stalk region (1500-2000 μm from HL) to identify spatially sequential molecular changes. Our findings showed significant overall gene expression variations among the head, proximal stalk, and distal stalk regions of pedunculated T1 CRCs compared to the control adenoma. Compared to LNM-negative T1 CRCs, LNM-positive T1 CRC showed that the expression of genes involved in immune-related pathways such as B2M, HLA-B, and HLA-E were significantly downregulated in the distal stalk region compared to the proximal stalk region. In summary, our results may tentatively suggest considering endoscopic resection of the stalk with a minimum 2000 μm margin from the HL, taking into account the gene expression alterations related to immune-related pathways. However, we acknowledge the limitations of this pilot study, notably the small case series, which may restrict the depth of interpretation. Further validation is imperative to substantiate these findings.

Spatial profiling of the placental chorioamniotic membranes reveals upregulation of immune checkpoint proteins during Group B Streptococcus infection in a nonhuman primate model.

Preterm birth is a leading cause of neonatal mortality, which is often complicated by intrauterine infection and inflammation. We have established a nonhuman primate model of Group B Streptococcus (GBS, Streptococcus agalactiae) infection-associated preterm birth. Immune checkpoints are modulators of the immune response by activating or suppressing leukocyte function and are understudied in preterm birth. The objective of this study was to spatially profile changes in immune protein expression at the maternal-fetal interface during a GBS infection with a focus on immune checkpoints. Twelve nonhuman primates (pigtail macaques, Macaca nemestrina) received a choriodecidual inoculation of either: 1) 1-5 X 108 colony forming units (CFU) of hyperhemolytic/hypervirulent GBS (GBSΔcovR, N=4); 2) an isogenic/nonpigmented strain (GBS ΔcovRΔcylE, N=4); or, 3) saline (N=4). A Cesarean section was performed at preterm labor or 3 days after GBS infection or 7 days after saline inoculation. Nanostring GeoMx® Digital Spatial Profiling technology was used to segment protein expression within the amnion, chorion, and maternal decidua at the inoculation site using an immuno-oncology panel targeting 56 immunoproteins enriched in stimulatory and inhibitory immune checkpoint proteins or their protein ligands. Statistical analysis included R studio, Kruskal-Wallis, Pearson and Spearman tests. Both inhibitory and stimulatory immune checkpoint proteins were significantly upregulated within the chorioamniotic membranes and decidua (VISTA, LAG3, PD-1, CD40, GITR), as well as their ligands (PD-L1, PD-L2, CD40L; all p<0.05). Immunostaining for VISTA revealed positive (VISTA+) cells, predominantly in the chorion and decidua. There were strong correlations between VISTA and amniotic fluid concentrations of IL-1β, IL-6, IL-8, and TNF-α (all p<0.05), as well as maternal placental histopathology scores (p<0.05). Differential regulation of multiple immune checkpoint proteins in the decidua at the site of a GBS infection indicates a major perturbation in immunologic homeostasis that could benefit the host by restricting immune-driven pathologies or the pathogen by limiting immune surveillance. Protein expression of VISTA, an inhibitory immune checkpoint, was upregulated in the chorion and decidua after GBS infection. Investigating the impact of innate immune cell expression of inhibitory immune checkpoints may reveal new insights into placental host-pathogen interactions at the maternal-fetal interface.

Abstract 5636: Multiplex digital spatial profiling (DSP) of proteins in the tumor microenvironment in response to GSK-3 inhibition by 9-ING-41 (elraglusib) correlates with novel immunostimulatory effects observed in vivo

Abstract Glycogen synthase kinase 3 (GSK-3) is a serine/threonine kinase with key roles in myriad biological processes such as tumor progression, and inhibition of GSK-3 using the small molecule elraglusib has shown promising preclinical antitumor activity in multiple tumor types. Our preclinical experiments showed that elraglusib treatment increased tumor cell PD-L1 expression, downregulated angiogenic and immunosuppressive signaling pathways, and increased anti-tumor immune responses in vitro and in vivo. Subsequent studies showed that several circulating factors were predictive of response to PD-1/PD-L1 blockade and GSK-3 inhibition in a murine model of colorectal cancer. To determine the translational relevance of our prior results we evaluated tumor biopsies and plasma samples from patients with refractory solid tumors of multiple tissue origins enrolled in a Phase 1 clinical trial investigating elraglusib (NCT03678883). Plasma samples were collected from patients at baseline and 24 hours post-IV administration of elraglusib and were analyzed using Luminex technology. Paired FFPE tumor biopsies from patients with colorectal or pancreatic cancer before and after treatment were selected to analyze the tumor microenvironment using NanoString GeoMx DSP technology. The region of interest (ROI) selection strategy focused on mixed tumor and immune cell segments and ROIs were segmented using panCK+ and CD45+ morphology stains. Cytokine analysis revealed that elevated baseline plasma levels of IL-1 beta and reduced levels of VEGF correlated with improved progression-free survival (PFS) and overall survival (OS). PFS was also found to be positively correlated with elevated plasma levels of immunostimulatory analytes such as Granzyme B, IFN-gamma, and IL-2 at 24 hours post-treatment with elraglusib. CD45+ tumor-infiltrating immune cells had lower expression of VISTA, PD-1, and IDO-1 inhibitory checkpoint proteins and higher expression of OX40L and B7-H3 stimulatory checkpoint proteins in post-treatment biopsies as compared to pre-treatment biopsies. Moreover, time-on-study length negatively correlated with CD39 expression in PanCK+ segments and positively correlated with CD163 expression in CD45+ segments. This ongoing study, to our knowledge, represents the first digital spatial analysis of tumor biopsies from patients treated with elraglusib. These novel circulating biomarkers of response to GSK-3 inhibition could provide significant clinical utility and the spatial proteomics data may give us insights into the immunomodulatory mechanisms of GSK-3 inhibition. Citation Format: Kelsey E. Huntington, Christoph Schorl, Shaolei Lu, Daniel Newhouse, Benedito A. Carneiro, Wafik S. El-Deiry. Multiplex digital spatial profiling (DSP) of proteins in the tumor microenvironment in response to GSK-3 inhibition by 9-ING-41 (elraglusib) correlates with novel immunostimulatory effects observed in vivo. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5636.

Spatial Analysis of Neural Cell Proteomic Profiles Following Ischemic Stroke in Mice Using High-Plex Digital Spatial Profiling

Stroke is ranked as the fifth leading cause of death and the leading cause of adult disability in the USA. The progression of neuronal damage after stroke is recognized to be a complex integration of glia, neurons, and the surrounding extracellular matrix, therefore potential treatments must target the detrimental effects created by these interactions. In this study, we examined the spatial cellular and neuroinflammatory mechanisms occurring early after ischemic stroke utilizing Nanostring Digital Spatial Profiling (DSP) technology. Male C57bl/6 mice were subjected to photothrombotic middle cerebral artery occlusion (MCAO) and sacrificed at 3 days post-ischemia. Spatial distinction of the ipsilateral hemisphere was studied according to the regions of interest: the ischemic core, peri-infarct tissues, and peri-infarct normal tissue (PiNT) in comparison to the contralateral hemisphere. We demonstrated that the ipsilateral hemisphere initiates distinct spatial regulatory proteomic profiles with DSP technology that can be identified consistently with the immunohistochemical markers, FJB, GFAP, and Iba-1. The core border profile demonstrated an induction of neuronal death, apoptosis, autophagy, immunoreactivity, and early degenerative proteins. Most notably, the core border resulted in a decrease of the neuronal proteins Map2 and NeuN; an increase in the autophagy proteins BAG3 and CTSD; an increase in the microglial and peripheral immune invasion proteins Iba1, CD45, CD11b, and CD39; and an increase in the neurodegenerative proteins BACE1, APP, amyloid β 1–42, ApoE, and hyperphosphorylated tau protein S-199. The peri-infarct region demonstrated increased astrocytic, immunoreactivity, apoptotic, and neurodegenerative proteomic profiles, with an increase in BAG3, GFAP, and hyperphosphorylated tau protein S-199. The PiNT region displayed minimal changes compared to the contralateral cortex with only an increase in GFAP. In this study, we showed that mechanisms known to be associated with stroke, such as apoptosis and inflammation, occur in distinct spatial domains of the injured brain following ischemia. We also demonstrated the dysregulation of specific autophagic pathways that may lead to neurodegeneration in peri-infarct brain tissues. Taken together, these data suggest that identifying post-ischemic mechanisms occurring in a spatiotemporal manner may lead to more precise targets for successful therapeutic interventions to treat stroke.

Spatially resolved whole transcriptome profiling in human and mouse tissue using Digital Spatial Profiling.

Emerging spatial profiling technology has enabled high-plex molecular profiling in biological tissues, preserving the spatial and morphological context of gene expression. Here, we describe expanding the chemistry for the Digital Spatial Profiling platform to quantify whole transcriptomes in human and mouse tissues using a wide range of spatial profiling strategies and sample types. We designed multiplexed in situ hybridization probes targeting the protein-coding genes of the human and mouse transcriptomes, referred to as the human or mouse Whole Transcriptome Atlas (WTA). Human and mouse WTAs were validated in cell lines for concordance with orthogonal gene expression profiling methods in regions ranging from ∼10-500 cells. By benchmarking against bulk RNA-seq and fluorescence in situ hybridization, we show robust transcript detection down to ∼100 transcripts per region. To assess the performance of WTA across tissue and sample types, we applied WTA to biological questions in cancer, molecular pathology, and developmental biology. Spatial profiling with WTA detected expected gene expression differences between tumor and tumor microenvironment, identified disease-specific gene expression heterogeneity in histological structures of the human kidney, and comprehensively mapped transcriptional programs in anatomical substructures of nine organs in the developing mouse embryo. Digital Spatial Profiling technology with the WTA assays provides a flexible method for spatial whole transcriptome profiling applicable to diverse tissue types and biological contexts.

Abstract 3806: High dimensional spatial gene expression analysis of tumor micro-environment in head and neck squamous cell carcinomas

Abstract Background: The complex and dynamic nature of the tumor-immune microenvironment (TME) presents challenges for identification of robust and predictive biomarkers in immuno-oncology (IO). Standard multiplex immunohistochemistry (mIHC) or multiplex immuno-fluorescent assays (mIF) allow for the phenotyping on the cellular level but are limited by the number of markers available and the detection systems. Gene expression or next generation sequencing (NGS) platforms provide high-plex bulk data sets about the TME, but the spatial context of the tumor and immune cell interactions is lost. The NanoString GeoMx Digital Spatial Profiler (DSP) combines spatial context, utilizing fluorescent morphological markers, with molecular profiling capabilities. This combination allows for the quantitative analysis of high-plex analyte abundance which can be traced back to a region of interest within a sample, providing biological inference in the region of interest. Methods: Formalin-fixed paraffin-embedded (FFPE) specimens from HNSCC patients were cut in 5µm sections for all analyses. Whole section tissue analysis for a 770 gene expression panel was previously performed utilizing the NanoString PanCancer IO 360 Gene Expression Panel. Specific tumor microenvironment (TME) region gene expression was analyzed using two different gene expression panels with the NanoString GeoMx digital spatial profiling technology, the GeoMx Immune Pathways Panel (84 genes) and the GeoMx Human Whole Transcriptome Atlas (WTA) panel (18,000 genes). For both panels, the TME regions of interest (ROI) were identified using a set of morphology markers consisting of panCK, CD45 and Syto13, 12 ROIs were selected from each sectioned tumor material. Results: The IO360 nCounter readout was high-throughput but lacked the spatial definition that can be crucial to understanding the heterogeneity of the TME. The GeoMx Immune Pathways Panel provided counts for 84 targets known to be crucial in the immuno-oncology field, including the PD-1/PD-L1 inhibitor pathway blockade. These counts were successfully mapped to the 12 ROIs on the tissue. The WTA panel provided an unbiased view of 18,000 protein-coding genes with the same ROI spatial resolution as the Immune Pathways Panel along with increased insight into the TME. Conclusions: The technologies described above enable the investigation of the TME for use in biomarker discovery in the field of immunotherapy but can also facilitate the discovery of prognostic markers or mechanism of action studies because of the high dimensional data with spatial context. The detailed analysis of the 18,000 genes provides exhaustive information on the biology of the tumor microenvironment that was formerly limited in spatial resolution. Citation Format: Carlee Hemphill. High dimensional spatial gene expression analysis of tumor micro-environment in head and neck squamous cell carcinomas [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 3806.

Spatially Resolved, Multiplexed Digital Characterization of Protein Distribution and Abundance in Formalin-Fixed, Paraffin-Embedded (FFPE) Diffuse Large B Cell Lymphoma Tissue Sections Based on the Nanostring® Digital Spatial Profiling (DSP) Technology

Imaging-based techniques are powerful tools for analyzing protein and mRNA expression and localization within the tumor microenvironment. However, they typically suffer from a number of challenges, including lack of dynamic range, difficult quantitation, and labor intensive workflow for very limited multiplexing. We have developed a novel platform based on the nCounter® barcoding technology that enables spatially resolved, digital characterization of proteins and mRNA in a highly multiplexed assay. The assay relies upon probes coupled to photocleavable oligonucleotide tags which are released from discrete regions of the tissue using focused through-objective UV (~ 365nm) exposure. An automated prototype capable of imaging, selective illumination using a digital mirror device, and sample collection was developed by modifying a standard microscope. Cleaved tags are quantitated in an nCounter assay, and counts are mapped back to tissue location, yielding a spatially-resolved digital profile of analyte abundance.To apply this technology to the characterization of hematological malignancies, we have developed the nCounter Vantage 3D™ Heme RNA panel that characterizes biology known to be relevant for these tumors, including specific profiling of MAPK, MYC signaling, NF-κB, PI3K-AKT, and B cell and T cell receptor signaling. We have also developed the Vantage 3D Heme panel of antibodies that focuses on common signaling pathways, with an emphasis on phosphorylated targets. We have used these panels to characterize differences in protein expression between ABC and GCB subtypes of diffuse large B cell lymphoma.3 ABC and 3 GCB lymphoma samples, previously identified by the NanoString Lymphoma Subtyping Test, were profiled on the DSP platform. 5 um tissue sections were stained with fluorescent antibodies for CD45 (to illuminate tumor and infiltrating immune cells), CD3 (to identify infiltrating T cells) and SYTO-83 (to illuminate nuclei), along with the oligo labeled antibodies from either the heme panel or the ST signaling panel. Regions of interested (ROI) were selected based on abundance of CD45 and CD3 staining from the ST-panel stained tissue section, and the same ROI was selected on the heme panel stained section using image alignment.In this study, we used these two Vantage 3D Heme panels to compare protein expression between the different subtypes of DLBCL and identify proteins that vary between subtype. We also identified heterogeneity of protein expression within the ABC subtype for the key tumor suppressors p53 and individual sample variation of expression in p-AKT within the GCB subtype. Furthermore, we observe strong concordance of signal from antibodies that are included in both antibody panels, suggesting technical reproducibility of the platform.Data are analyzed on software that is being developed in parallel with the technology, which enables visualization of the individual ROI within a sample, comparison of ROI across samples, and comparison of the individual probe counts within and between ROI.ConclusionsNanostring digital spatial profiling provides a quantitative and reproducible digital readout for multiplexed protein expression from the same microscopic regions of individual tissue sections. In this study we demonstrate how DSP technology can help elucidate protein expression profiles within individual lymphoid tumor specimens. First we show how regions of interest with the tumor (ROI) are identified and profiled using two multiplex protein panels simultaneously. Second, we demonstrate high platform concordance between proteins present within different NanoString panel products. Finally, we present methodology and data analysis workflow capable of identifying protein expression differences both within individual tumors, and between ABC and GCB lymphoma subtypes. Application of this technology to the study of hematological malignancies may help to identify novel drug targets and/or characterize response to therapy, thus helping to define novel biology and accelerate the clinical development path. DisclosuresWhite:Nanostring Technologies Inc.: Employment. Elliott:Nanostring Technologies Inc.: Employment. Liang:Nanostring Technologies Inc.: Employment. Warren:NanoString Technologies: Employment, Equity Ownership. Beechem:NanoString Technologies: Employment, Equity Ownership.

Advanced Molecular Characterization Using Digital Spatial Profiling Technology on Immunooncology Targets in Methylated Compared with Unmethylated IDH-Wildtype Glioblastoma

Introduction Glioblastoma (GBM) is the most common primary adult brain tumour with a median overall survival (OS) of 12–15 months. Molecular characterization of multiple immunooncology targets in GBM may help target novel immunotherapeutic strategies. We used NanoString GeoMx® Digital Spatial Profiling (DSP) to assess multiple immunooncology protein targets in methylated versus unmethylated IDH-wild-type glioblastoma. Methods NanoString GeoMx® DSP technology uses multiple primary antibodies conjugated to indexing DNA oligos with a UV photocleavable linker. Tissue regions of interest (ROIs) are selected with bound fluorescent antibodies; oligos are released via a UV-mediated linker and quantitated. We used DSP multiplex analysis of 31 immunooncology proteins and controls (CD4, CD14, CD68, CD8A, B7-H3, PD-L1, CD19, FOXP3, CD44, STAT3 (phospho Y705), CD45, Pan Cytokeratin, MS4A1/CD20, CD45RO, PD1, CD3, beta-2 microglobulin, VISTA, Bcl2, GZMB, PTEN, beta-catenin, CD56, Ki-67, STAT3, AKT, p-Akt, S6, Histone H3, IgG Rabbit control, and Mouse IgG control) from ROIs in a cohort of 10 IDH-wild-type glioblastomas (5 methylated and 5 unmethylated). An nCounter platform allowed quantitative comparisons of antibodies between ROIs in MGMT methylated and unmethylated tumours. Mean protein expression counts between methylated and unmethylated GBM were compared using technical and biological replicates. Results The analysis showed 10/27 immunooncology target proteins were significantly increased in methylated versus unmethylated IDH-wild-type glioblastoma tumour core (false discovery rate (FDR) <0.1 by Benjamini–Hochberg procedure). Conclusions NanoString GeoMx® DSP was used to analyse multiple immunooncology protein target expression in methylated versus unmethylated IDH-wild-type glioblastoma. In this small study, there was a statistical increase in CD4, CD14, CD68, CD8A, B7-H3, PDL-1, CD19, FOXP3, CD44, and STAT3 protein expression in methylated versus unmethylated GBM tumour core; however, this requires larger cohort validation. Advanced multiplex immunooncological biomarker analysis may be useful in identifying biomarkers for novel immunotherapeutic agents in GBMs.

Spatially-resolved proteomics and transcriptomics: An emerging digital spatial profiling approach for tumor microenvironment

Digital spatial profiling (DSP) is an emerging powerful technology for proteomics and transcriptomics analyses in a spatially resolved manner for formalin-fixed paraffin-embedded (FFPE) samples developed by nanoString Technologies. DSP applies several advanced technologies, including high-throughput readout technologies (digital optical barcodes by nCounter instruments or next generation sequencing (NGS)), programmable digital micromirror device (DMD) technology, and microfluidic sampling technologies into traditional immunohistochemistry (IHC) and RNA in situ hybridization (ISH) approaches, creating an innovative tool for discovery, translational research, and clinical uses. Since its launch in 2019, DSP has been rapidly adopted, especially in immuno-oncology and tumor microenvironment research areas, and has revealed valuable information that was inaccessible before. In this article, we report the successful setup and validation of the first DSP technology platform in China. Both DSP spatial protein and RNA profiling approaches were validated using FFPE colorectal cancer tissues. Regions of interest (ROIs) were selected in the areas enriched with tumor cells, stroma/immune cells, or normal epithelial cells, and multiplex spatial profiling of both proteins and RNAs were performed. DSP spatial profiling data were processed and normalized accordingly, validating the high quality and consistency of the data. Unsupervised hierarchical clustering as well as principal component analysis (PCA) grouped tumor, stroma/immune cells, and normal epithelial cells into distinct clusters, indicating that the DSP approach effectively captured the spatially resolved proteomics and transcriptomics profiles of different compartments within the tumor microenvironment. In summary, the results confirmed the expected sensitivity and robustness of the DSP approach in profiling both proteins and RNAs in a spatially resolved manner. As a novel technology in highly complex spatial analyses, DSP endows refined analytical power from the tumor microenvironment perspective with the potential of scaling up to more analyzable targets at relatively low cell input levels. We expect that the DSP technology will greatly advance a wide range of biomedical research, especially in immuno-oncology and tumor microenvironment research areas.

Abstract 840: Pathway enrichment analysis of gene expression data from formalin-fixed paraffin embedded (FFPE) samples using the GeoMx™ DSP Platform

Abstract The GeoMx Digital Spatial Profiling (DSP) Platform enables robust detection of high-plex protein and RNA expression from user-defined sections within FFPE samples. As the number of targets detected within such tissues increases, it becomes important to apply systems biology strategies in order to better interpret the complex biology of the tumor microenvironment. In this study, we investigated the expression profiles of more than 1600 genes by utilizing more than 10,000 DSP-specific in situ hybridized (ISH) probes on FFPE samples. The bioinformatics tools we have development are enabling us to move beyond single-gene profiling to a better understanding of pathway-based expression. Our study includes colorectal cancer patient samples from which we have matching bulk RNA sequencing and NanoString analysis to compare. The panel of genes profiled have a strong focus around capturing biological signaling along canonical signaling pathways and cell-intrinsic signaling from immune cells and other cell types. We demonstrate the ability to leverage foundational pathway interrogation tools, including Reactome, with the data to capture spatially-resolved pathway interactions and signaling within FFPE tissues. As we look towards the future of the GeoMx platform and high-plex RNA profiling of tissue samples, these experiments highlight not only the need but the capacity for this platform to derive deep understanding of the biology within and across a single slide of tissue. These experiments are being used to drive development of the software features within the GeoMx ecosystem, which will provide further support for pathway-level exploration of expression when working with highly multiplexed reagents in future platform offerings. GeoMx™ DSP technology is for Research Use Only and not for use in diagnostic procedures. Citation Format: Tressa R. Hood, Jason Reeves, Zach Norgaard, Margaret Hoang, Sarah Warren, Erin Piazza, Rich Boykin, Joseph Beechem. Pathway enrichment analysis of gene expression data from formalin-fixed paraffin embedded (FFPE) samples using the GeoMx™ DSP Platform [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 840.

Advanced molecular characterization using Digital Spatial Profiling Technology on immuno-oncology targets in methylated compared with unmethylated IDH-wildtype glioblastoma

Abstract Introduction Glioblastoma (GBM) is the most common primary adult brain tumour with 12–15 months median overall survival. Molecular characterization of multiple immuno-onology targets in GBM is required to optimise immunotherapeutic strategies. Advanced molecular characterisation using Digital Spatial Profiling Technology allows assessment of multiple immuno-oncology protein targets in methylated compared with unmethylated Glioblastoma IDH-wildtype. Methods Nanostring DSP uses multiple primary antibodies conjugated to indexing DNA oligos with a UV photocleavable linker. Tissue Regions of interest (ROI) are selected with bound fluorescent antibodies, oligos are released via a UV-mediated linker and quantitated. We used DSP multiplex analysis of 31 immuno-oncology proteins and controls (PD-1, PD-L1, B7-H3, VISTA, CD45, CD45RO, CD3, MS4A1 (CD20), CD4, CD8A, CD68, STAT3, STAT3 (Phopshory705), GZMB, Beta-2-microglobulin, CD56, Beta-catenin, FOXP3, CD14, CD19, AKT, P-AKT, PTEN, Bcl-2, CD44, Histone H3, S6, IgG Rabbit Control, Mouse IgG Control, Pan-Cytokeratin, Ki67) from ROIs in a cohort of 10 Glioblastomas IDH-wildtype (5 methylated, 5 unmethylated). An nCounter platform allowed quantitative comparisons of antibodies between ROIs in MGMT methylated and unmethylated tumours. Mean protein expression counts between methylated and unmethylated GBM were compared using a linear mixed effect model for technical and biological replicates. Results GeoMx DSP showed 10/27 immuno-oncology target proteins were significantly increased in methylated versus unmethylated Glioblastoma IDH-wildtype (false discovery rate FDR &lt;0.1 by Benjamini-Hocheberg Procedure). Conclusions Increased immuno-oncology protein target expression in methylated versus unmethylated glioblastoma IDH-wildtype using DSP platform has been identified. Advanced multiplex immuno-oncological biomarker analysis is required to identify predictive biomarkers for novel immunotherapeutic agents in GBMs.

High-Plex Predictive Marker Discovery for Melanoma Immunotherapy-Treated Patients Using Digital Spatial Profiling.

Protein expression in formalin-fixed, paraffin-embedded tissue is routinely measured by IHC or quantitative fluorescence (QIF) on a handful of markers on a single section. Digital spatial profiling (DSP) allows spatially informed simultaneous assessment of multiple biomarkers. Here we demonstrate the DSP technology using a 44-plex antibody cocktail to find protein expression that could potentially be used to predict response to immune therapy in melanoma.Experimental Design: The NanoString GeoMx DSP technology is compared with automated QIF (AQUA) for immune marker compartment-specific measurement and prognostic value in non-small cell lung cancer (NSCLC). Then we use this tool to search for novel predictive markers in a cohort of 60 patients with immunotherapy-treated melanoma on a tissue microarray using a 44-plex immune marker panel measured in three compartments (macrophage, leukocyte, and melanocyte) generating 132 quantitative variables. The spatially informed variable assessment by DSP validates by both regression and variable prognostication compared with QIF for stromal CD3, CD4, CD8, CD20, and PD-L1 in NSCLC. From the 132 variables, 11 and 15 immune markers were associated with prolonged progression-free survival (PFS) and overall survival (OS). Notably, we find PD-L1 expression in CD68-positive cells (macrophages) and not in tumor cells was a predictive marker for PFS, OS, and response. DSP technology shows high concordance with QIF and validates based on both regression and outcome assessment. Using the high-plex capacity, we found a series of expression patterns associated with outcome, including that the expression of PD-L1 in macrophages is associated with response.

Abstract 4691: Evaluation of the NanoString’s Digital Spatial Profiling (DSP) technology in formalin-fixed paraffin embedded (FFPE) cell line mixtures, PBMCs and non-small cell lung cancer (NSCLC) tissues

Abstract Evaluation of the NanoString’s Digital Spatial Profiling (DSP) Technology in Formalin-Fixed Paraffin Embedded (FFPE) cell line mixtures, PBMCs and Non-Small Cell Lung Cancer (NSCLC) tissues Purpose: The complex nature of the tumor requires multiplexed, quantitative analysis of tissues which is challenging using immunohistochemistry (IHC). Several novel approaches (including NanoString Digital Spatial Profiling (DSP) Technology) were developed to enable multiplexed analysis of tissues. Here we report on a comparative evaluation of the GeoMx™ DSP platform including a comparison to IHC. Methods: Performance of DSP methodology was assessed using Raji and CEM/C1 cell line mixtures, as well as healthy donor PBMCs that had been evaluated using flow cytometry for expression of the targeted markers: CD4, CD8, CD68, CD14, CD3, CD45, CD45RO, CD20 and GZMB. DSP technology was compared with IHC by measuring T-cells markers (CD3, CD4, CD8), a monocytic marker (CD68) as well as PD-L1 expression in five FFPE NSCLC tumor specimens. Serial sections were stained by IHC, hematoxylin and eosin (H&amp;E) and regions of interests (ROIs) were selected to sample areas with varied expression of each of the markers (qualified as low, medium and high). Selected ROIs were then assayed on the GeoMx DSP platform in a blinded fashion using a 39-plex immune panel that included CD3, CD4, CD8, CD68 and PD-L1 antibodies and DSP counts were compared to qualitative IHC scoring. Results: Cell line mixture experiments demonstrated that protein counts were highly correlated to the fractions of cell lines expressing a given marker (r=0.90 - 0.94). The assay also showed good concordance with flow cytometry measurements (r=0.49-0.51 between flow cytometry MFIs and NanoString counts for PMBCs). DSP counts related well to the IHC staining patterns, as higher DSP measurements were detected in ROIs classified as high based IHC staining intensity for the markers investigated. As expected, counts were lower for markers such as CD8 and PD-L1 that were also classified as low expressing by IHC in the analyzed NSCLC tumor samples. Exploratory analysis of co-expression of all 39 markers included in the DSP panel in various areas of the tumors suggested that DSP technology can be used to perform detailed characterization of various areas of the tumor. Conclusions: Our evaluation affirms that NanoString’s GeoMx DSP platform is a promising option for multiplexed analysis of tumor tissues. DSP technology produced measurements comparable to those obtained with flow cytometry and IHC for the evaluated markers. These data show that DSP technology can collect high-parametric proteomics data from FFPE tissue samples which can help to study the complex biology of tumor microenvironment. Citation Format: Joshua James Rusbuldt, Tanesha Cash-Mason, Shaozhou Tian, Alison VanSchoiack, Yan Liang, Chandra Rao, Denis Smirnov. Evaluation of the NanoString’s Digital Spatial Profiling (DSP) technology in formalin-fixed paraffin embedded (FFPE) cell line mixtures, PBMCs and non-small cell lung cancer (NSCLC) tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4691.

Abstract 499: NanoString®GeoMx®digital spatial profiling further defines the role of B cells in the response to immune checkpoint blockade

Abstract Background: While treatment with immune checkpoint blockade (ICB) has markedly improved outcomes in advanced melanoma patients and other malignancies, predicting response remains a challenge. Biomarkers including tumor mutational burden (TMB), T-cell infiltration, and PD-L1 expression, have been identifiedbut remain inadequate. Other components of innate and adaptive immunity, including B-cells and tertiary lymphoid structures (TLS), have been implicated in the response to other cancer therapies, andpreclinical data suggests B cells may contribute in the response to immunotherapy. Here, we use targeted protein expression profiling-via NanoString digital spatial profiling (DSP) technology (research use only)-to demonstrate a role for B cells in the response to ICB in patients with high-risk resectable melanoma; furthermore, we characterize the B cell subsets that enable this function. Methods: We conducted a phase 2 clinical trial of neoadjuvant ICB therapy in patients with high-risk resectable melanoma (PD-1 blockade monotherapy or combined CTLA-4/PD-1 blockade) (NCT02519322). Longitudinal tumor samples were taken during therapy. Formalin-fixed paraffin embedded tissue sections from tumor samples (n=10 responders [R], n=10 non-responders [NR]) were analyzed by NanoString DSP technology and stained with a cocktail of S100B, CD45, CD19 and a 40-protein cocktail of antibodies conjugated to UV-photocleavable DNA barcodes. Regions of interest (ROI) were delineated using immunofluorescence followed by UV excitation of the defined ROIs, releasing the DNA barcodes for downstream quantitation on the NanoString nCounter®platform. Utilizing masking strategies, we define the unique expression pattern within discrete subsets of immune cells. These same tumors have concurrently been analyzed by RNAseq, immunohistochemistry, CYTOF, and single-cell RNAseq. Results: Biomarker counts are highly concordant across samples from the same patient’s tumor. High concordance between DSP and quantitative fluorescence is seen as a validation for the DSP method. We identify B cells as components of TLS; the B cells are closely integrated with CD4 and CD8 T cells and follicular dendritic cells. There are significantly more TLS identified in R compared to NR patients. Utilizing CYTOF, we concurrently identify specific subsets of B cells present within the TLS associated with response. We characterize the spatial relationship of these same B cell subsets with other components of the TLS and define the protein expression patterns of these cells. Conclusion: NanoString DSP data complement our deep molecular and immune profiling of tumors from melanoma patients treated with ICB; together, they provide a novel predictive role for B-cells and TLS in the response to ICB and, importantly, provide mechanistic insight into their potential contribution in the response to cancer therapy. Citation Format: Beth A. Helmink, Sangeetha Reddy, Jorge Blando, Yan Liang, Sarah Warren, Vancheswaran Gopalakrishnan, Hussein A. Tawbi, Rodabe N. Amaria, Michael Davies, Jeffrey E. Gershenwald, Elizabeth Burton, Rafet Basar, Alexander J. Lazar, Courtney W. Hudgens, Katy Rezvani, James P. Allison, Padmanee Sharma, Joseph M. Beechem, Jennifer A. Wargo, Michael T. Tetzlaff. NanoString®GeoMx®digital spatial profiling further defines the role of B cells in the response to immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 499.

New tools for pathology: a user's review of a highly multiplexed method for in situ analysis of protein and RNA expression in tissue.

Tumor cell heterogeneity and tumor cell-stromal interactions are being explored as determinants of disease progression and treatment resistance in solid tumor and hematological malignancies. As such, tools simultaneously capable of highly multiplexed profiling of tissues' protein and RNA content, as well as interrogation of rare or single cells, are required to precisely characterize constituent tumor cell populations, infiltrating lymphocytes and stromal elements. Access to spatial relationships will enable more precise characterization of tumors, support patient stratification and may help to identify novel drug targets. Multiple platforms are being developed to address these critical unmet needs. The NanoString digital spatial profiling (DSP) platform enables highly multiplexed, spatial assessment of protein and/or RNA targets in tissues by detecting oligonucleotide barcodes conjugated via a photocleavable linker to primary antibodies or nucleic acid probes. Although this platform enables high-dimensional spatial interrogation of tissue protein and RNA expression, a detailed understanding of its composition, function and chemistry is advisable to guide experimental design and data interpretation. The purpose of this review is to provide an independent, comprehensive description of the DSP technology, including an overview of NanoString's capture and antibody barcode conjugation chemistries, experimental workflow, data output and analysis methods. The DSP technology will be discussed in the context of other highly multiplexed immunohistochemistry methods, including imaging mass cytometry and multiplexed ion beam imaging, to inform potential users of the advantages and limitations of each. Additional issues such as preanalytical variability, sampling and specimen adequacy will be considered with respect to the platforms to inform potential experimental design. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Abstract 3858: Validation of digital spatial profiling of key immuno-oncology targets for mouse FFPE preclinical models

Abstract Mouse models of cancer are an invaluable biomedical research tool in which characterization of the abundance, distribution and co-localization of immuno-oncology targets is critical for the advancement of disease understanding. Nanostring Technologies' Digital Spatial Profiling (DSP) technology allows for the simultaneous analysis of 10s to 100s of proteins from discrete regions of interest (ROI), providing a spatial context to multiplexed data. The purpose of this study is to validate an antibody panel designed to characterize key tumor and immunology markers on the DSP platform. DSP technology utilizes barcoded oligo tags that are covalently linked to detection reagents, in this case, antibodies. The workflow for a typical DSP experiment consists of incubating a formalin-fixed, paraffin-embedded (FFPE) tissue with a cocktail of oligo-labeled primary antibodies and three fluorescently labeled visualization markers. The visualization markers and tissue morphology guide the selection of ROIs. UV light then illuminates each ROI, releasing the oligo tags through a UV photocleavable linker. Tags are collected through microcapillary aspiration and then deposited into a microtiter plate for further analysis on the standard nCounter® platform. In order to validate antibodies for this technology, the specificity and sensitivity of each antibody was evaluated for proper immunohistochemistry (IHC) staining pattern on appropriate tissues as well as signal-to-noise ratios of positive counts above background. Panels will also undergo an evaluation of single vs. multiplex interactions to ensure there are no deleterious effects of multiplexing antibodies. Immunohistochemical analysis of antibodies chosen for panel displays indistinguishable staining patterns on mouse control tissues for unconjugated primary antibodies and oligo-conjugated primary antibodies. Visualization markers allow for an ROI selection strategy that utilizes a range of circular ROIs (e.g., 50-650um) as well as custom masking of areas of interest. Key immuno-oncology targets including B7-H3, beta-catenin, CD19, CD25, CD3, CD4, histone H3, Ly-6G, pan-cytokeratin, PD-1, PD-L1, F4/80, Ki-67, STAT3, etc., have been validated thus far. Ongoing validation efforts will continue to expand this antibody panel. Nanostring Technologies' DSP platform is applicable not only to human tissue but also to mouse preclinical samples. Antibody specificity, sensitivity and interaction within a cocktail have been validated for this application. DSP technology allows for the multiplexing of important mouse immuno-oncology markers so that researchers can evaluate the abundance as well as the spatial distribution of these key targets simultaneously. Citation Format: Sarah Warren, Heather Metz, Kristi Barker, JingJing Gong, Alison VanSchoiack, Quoc Huynh, Chris Merritt, Lucas Dennis, Yan Liang, Joseph M. Beechem. Validation of digital spatial profiling of key immuno-oncology targets for mouse FFPE preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3858.

Abstract 3621: High-plex immune marker spatial profiling quantitation by NanoString Digital Spatial Profiling technology and quantitative immunofluorescence

Abstract Background: Quantitative immunofluorescence (QIF) offers the advantage of multiple target measurement on a single slide, but is limited by the number of fluorescence channels. NanoString's Digital Spatial Profiling (DSP)* can detect and quantify immune markers at higher multiplex with spatial resolution within specific regions of interest on FFPE tissue. Here, we compare NanoString DSP to automated QIF (AQUA), for immune marker compartment specific measurement. Additionally, we assess their agreement on the prognostic value of commonly used biomarkers in Non-Small Cell Lung Cancer (NSCLC). Finally, we explore the predictive value of a 30-plex panel of immune markers on two cohorts of treated patients. Methods: NanoString DSP technology uses a cocktail of primary antibodies conjugated to indexing DNA oligos. Regions of interest (ROI) on the tissue are selected with fluorescently labeled antibodies, and oligos from that region are UV cleaved and quantified on the nCounter platform. The comparator for this technology was the AQUA method of QIF. We retrospectively examined a NSCLC cohort of over 100 patients for prognostic markers and two treated patient cohorts for associations between immune markers expression and response to treatment, all in tissue microarray format. The treated cohorts included an Epidermal Growth Factor Receptor Tyrosine kinase inhibitor (EGFR TKI) treated NSCLC cohort and a melanoma immunotherapy (ITx) treated cohort. Results: Multiple immune markers (CD3, CD4, CD8, CD20, PD-L1) were assessed and a high correlation was found between NanoString DSP counts and QIF scores, when the measurements were performed in the same ROIs (tumor or stroma). The prognostic value of the immune markers tested was concordant between the two assays with high expression of CD3 assessed by either assay showing a statistically significantly better overall survival (OS). For the EGFR TKI treated NSCLC cohort none of the immune markers was correlated with response to treatment or favorable outcome. However, in the ITx treated melanoma patients, PD-L1, PD-1, CD68, CD3, CD8A and b2-microglobulin tumor expression predicted response to treatment. Each marker also predicted better outcome, but only CD8A was an independent predictive marker of prolonged survival. Conclusion: NanoString DSP offers the capacity of highly multiplexed immune marker measurements on selected compartments. It shows high concordance with AQUA, which was further validated by comparing prognostic significance. The pilot study of the Melanoma ITx cohort also illustrates the potential to simultaneously evaluate a range of markers and possibly construct new predictive signatures based on a cohort represented by very small tissue samples. *FOR RESEARCH USE ONLY. Not for use in diagnostic procedures. Citation Format: Maria I. Toki, Pok Fai Wong, Harriet Kluger, Yuting Liu, Chris Merritt, Giang Ong, Sarah Warren, Joseph M. Beechem, David L. Rimm. High-plex immune marker spatial profiling quantitation by NanoString Digital Spatial Profiling technology and quantitative immunofluorescence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3621.

Abstract 3955: Spatially resolved, multiplexed digital characterization of protein and mRNA distribution and abundance in formalin-fixed, paraffin-embedded (FFPE) tissue sections based on NanoString’s Digital Spatial Profiling (DSP) technology: applications to immuno-oncology (IO) and tumor heterogeneity

Abstract Introduction: As intra-tumoral heterogeneity has emerged as a challenge in development of targeted cancer therapeutics, the tissue context of biomarker levels and colocalization of key immunoregulatory proteins has become an increasingly important aspect for understanding tumor immune responses, patient classification, and stratification. Historically, immunohistochemistry and in situ hybridization have been used to assess spatial heterogeneity of targets in clinical samples. These approaches, however, have limited multiplexing capacity and dynamic range. Here, we use DSP technology, a spatially resolved approach for quantifying up to 800 protein or RNA targets with over 5 logs of dynamic range in a single FFPE slide to overcome these limits. Methods: The technology uses DNA oligo tags for either protein or RNA detection. For protein detection, a cocktail of 30+ primary antibodies (Abs), each with a unique, photocleavable oligo tag, and 1-3 fluorescently (FL) labeled Abs was applied to a slide-mounted FFPE tissue section. Regions of interest (ROI), selected based on a FL imaging scan of the entire tissue, were illuminated sequentially with focused UV light to release the oligos. Following each UV cycle, eluent was collected from the local ROI, moved to a microtiter plate, hybridized to NanoString® barcodes, and then analyzed with an nCounter® Analysis System. The resulting digital counts corresponded to the abundance of each targeted protein in the ROI. For RNA detection, a cocktail of multiple UV-cleavable in situ hybridization probes were used in a similar manner. Results: We demonstrate multiplexed detection from discrete ROIs within a tumor and adjacent normal tissue, enabling systematic interrogation of a heterogeneous tumor microenvironment. In control samples, we found expected levels of protein and RNA targets. We further demonstrate that this approach enables analysis of target abundance from individually selected cells, both contiguous and non-contiguous with the same phenotype, and enables multiplexed detection of key IO targets. Finally, we demonstrate detection of key IO RNA targets using direct hybridization of oligo-labeled probes. Conclusions: With further development, our novel DSP approach to capture the spatial context of protein and RNA levels will have many applications in biomarker and translational research. The ability to digitally measure RNA and protein at up to 800-plex from FFPE tissues could facilitate drug mechanism-of-action and response studies within the tumor microenvironment. Quantitative, high-plex data should also greatly accelerate the discovery of IO biomarkers in tumors and the development of companion diagnostics for targeted therapies. Citation Format: Chris Merritt, Jaemyeong Jung, Giang Ong, Yan Liang, Fiona Pakiam, Dwayne Dunaway, Isaac Sprague, Sarah Warren, Gordon B. Mills, Joseph Beechem. Spatially resolved, multiplexed digital characterization of protein and mRNA distribution and abundance in formalin-fixed, paraffin-embedded (FFPE) tissue sections based on NanoString’s Digital Spatial Profiling (DSP) technology: applications to immuno-oncology (IO) and tumor heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3955. doi:10.1158/1538-7445.AM2017-3955

Abstract 3810: Validation of novel high-plex protein spatial profiling quantitation based on NanoString's Digital Spatial Profiling (DSP) technology with quantitative fluorescence (QIF)

Abstract Background: Protein expression in formalin-fixed, paraffin-embedded (FFPE) tissue is routinely measured by Immunohistochemistry (IHC) on only one protein, or with quantitative fluorescence (QIF) on a handful of proteins on a single section. NanoString’s Digital Spatial Profiling (DSP)* technology can detect and quantify proteins at significantly higher multiplex (currently 30 plex, potential for up to 800 plex) with spatial resolution using molecular digital color-coded “barcodes”, within specific regions of interest on FFPE tissue. Here, we compare Nanostring DSP technology to automated QIF (AQUA), for accurate and reproducible measurement of protein expression on a continuous scale. Additionally, using the multiplexing potential of Nanostring technology, we did a pilot study to assess cold ischemic time as a variable to monitor tissue quality by assessment of epitope degradation in Non-Small Cell Lung Cancer (NSCLC). Methods: The DSP technology uses a cocktail of primary antibodies conjugated to indexing DNA oligos with a UV Photocleavable linker that can be counted on the nCounter™ platform. Regions of interest (ROI) on the tissue are selected with fluorescently labeled antibodies, and oligos from that region are released via UV mediated linker cleavage. Free oligos are captured via microcapillary fluidics into a microtiter plate and then quantitated on the nCounter. The comparator for this technology was the AQUA method of QIF. We examined a breast cancer tissue microarray with a range of HER2 expression, and a NSCLC time to fixation standardization array with timepoints from 20 min to 48 hrs. Results: Multiple markers were assessed and a high correlation was found between Nanostring DSP counts and QIF scores (R²&amp;gt;0.7), when the measurements were performed in the same region of interest (defined by cytokeratin expression). The dynamic range of DSP exceeded the quantification range of QIF (nearly 4 logs vs about 2 logs). When the 28 protein markers’ expression was compared at different fixation timepoints, most were found to be stable over different cold ischemic timepoints. Two markers, including phospho-ERK (Extracellular Signal-Regulated Kinases) and phospho-GSK (Glycogen Synthase Kinase) showed epitope loss as a function of delay to fixation (R²=0.0064 and R²=0.05 respectively). Conclusion: The Nanostring DSP is a reproducible method with the capability of highly multiplexed measurements of protein expression on a field of view averaged basis. It shows high concordance with the AQUA method of QIF, an extensively validated technique for protein quantification. For the first time, the high-plex capacity of DSP allows inclusion of markers that are sensitive to time to fixation as an intrinsic control for tissue quality. *FOR RESEARCH USE ONLY. Not for use in diagnostic procedures. Citation Format: Maria I. Toki, Chris Merritt, Giang Ong, Joseph M. Beechem, Daniel E. Carvajal-Hausdorf, Yalai Bai, David Rimm. Validation of novel high-plex protein spatial profiling quantitation based on NanoString's Digital Spatial Profiling (DSP) technology with quantitative fluorescence (QIF) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3810. doi:10.1158/1538-7445.AM2017-3810