Chris Merritt

Abstract 5350: A comprehensive and integrated approach to genomic and proteomic analysis of FFPE NSCLC tumor specimens

This poster describes a novel fully integrated method for the multiplexed DNA, RNA and Protein analysis of formalin fixed paraffin embedded tumor specimens. Cancer progression is typically a result of aberrations in the molecular pathways that regulate cell growth. Identifying and understanding these changes is necessary for the detection of drug targets as well as the identification of novel biomarkers that can predict response to such drugs. Deep molecular profiling of clinical tumor samples is challenged by limited material and the compromised quality resulting from variable handling and the fixation processes that are commonly employed in the clinical laboratories. While multiplexed gene expression and mutational analysis of such samples has become well established, we describe a new method that enables multiplexed DNA, RNA and protein analysis from a single formalin fixed paraffin embedded (FFPE) tumor specimen on the NanoString nCounter system. We have adapted the sample preparation methods to enable the multi-analyte analysis of FFPE samples and have demonstrated concordance with the clinical gold standard immunohistochemistry (IHC) methodology. As a proof-of-concept, five FFPE non-small cell lung cancer (NSCLC) specimens were simultaneously stained with a panel of antibodies specific to a variety of cancer-relevant proteins, five of which were validated on serial sections by IHC using antibodies from Cell Signaling Technology. In addition to the protein analysis, from the same specimen, expression of 770 cancer-related genes and >100 single nucleotide variants (SNVs) were also analyzed on the nCounter platform. By integrating the analysis of DNA SNV’s, as well as RNA and protein expression from single FFPE specimens, it is now possible to reveal the molecular mechanisms of tumor progression, uncover novel drug targets and identify biomarkers from precious specimens. Citation Format: Douglas Hinerfield, Jennifer Mellen, Gary Geiss, Philippa Webster, Chris Merritt, Kristi Barker, Gokhan Demirkan, Brian Filanoski, Roberto Polakiewicz, Katherine Crosby. A comprehensive and integrated approach to genomic and proteomic analysis of FFPE NSCLC tumor specimens [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 5350. doi:10.1158/1538-7445.AM2017-5350

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

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 B081: Spatially-resolved, multiplexed (up to 800 plex) digital characterization of protein and mRNA abundance in FFPE tissue sections: Application to immuno-oncology

The Tumor Microenvironment (TME) has emerged as a key compartment that determines the overall effectiveness of cancer immunotherapy. Hence, it is very important to determine the abundance and location of key immune-regulators in the TME. Historically, standard immunohistochemistry (IHC) and immunofluorescence have been used to assess spatial heterogeneity of proteins and nucleic-acids in tissue slices. However, these techniques are inherently limited in utility because it has been difficult to quantify the abundance of multiple protein/nucleic-acids across a wide dynamic range. Here, we report the development and validation of a spatially-resolved protein and RNA detection platform with the potential to simultaneously quantify up to 800 targets with greater than 5 log10 of dynamic range from a single formalin-fixed paraffin-embedded (FFPE) slide. We demonstrate validation of this technology by characterization of a panel of immune proteins expressed in colorectal cancer samples, and we also demonstrate spatially resolved detection of RNA. In situ high-plex digital molecular profiling is enabled by the use of UV-photocleavable small indexing DNA-oligo tags that can be delivered to the target within the tissue via direct attachment to RNA binding probes or conjugation to primary antibodies and are quantified with the standard nCounter technology. A slide-mounted FFPE tissue section is bound with a multiplexed cocktail of oligo-labeled primary antibody or mRNA hybridization probes, and a microfluidic flow cell is attached to the slide. Low-plex (3 or 4 color fluorescence) visible wavelength probes are utilized to generate an overall view of the FFPE tissue slice morphology (e.g., nuclear staining probes, select antibody pairs such as anti-CD3 and anti-CD8). Using the visible wavelength morphology as a guide, regions of interest (ROI) in the tumor are identified (e.g., areas with tumor infiltrating leukocytes) and then sequentially illuminated with UV light to release the indexing-oligos off all the high-plex molecular profiling reagents. Using this approach and standard microscope instrumentation, the limits of detection enable near single cell resolution. Following each UV illumination cycle, the photocleaved indexing-oligos are released into the buffer-layer above the tissue slice, collected via microcapillary aspiration, and stored in an individual well of a microtiter-plate. The contents of each well can then be referenced back to the exact region of tumor that was illuminated by UV light. Oligos are then hybridized to the nCounter fluorescently labeled optical barcodes to permit ex-situ digital counting of as many as 800 different analytes localized within a single ROI in the tumor. As demonstration of the technology, simultaneous multiplexed detection of CD3, CD8, CD45R0, CD4, CD45, PD-1, PD-L1, Vista, TIM-3, B7-H3, Ki67 (plus additional key IO-targets) will be quantified from colorectal tumor biopsies using oligo-conjugated primary antibodies. Furthermore, we will demonstrate detection of key IO associated immune RNA targets using direct hybridization of oligo-labeled probes. The ability to measure DNA, RNA, and protein at up-to 800-plex from single slices of FFPE tissue may enable the discovery of key immune biomarkers in tumors and accelerate the development immunotherapy and their associated companion diagnostics. Citation Format: Dwayne Dunaway, Jaemyeong Jung, Chris Merritt, Isaac Sprague, Philippa Webster, Sarah Warren, Joseph Beechem. Spatially-resolved, multiplexed (up to 800 plex) digital characterization of protein and mRNA abundance in FFPE tissue sections: Application to immuno-oncology [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B081.

Abstract 1371: Spatially-resolved, multiplexed digital characterization of protein distribution and abundance in FFPE tissue sections

Intratumoral heterogeneity has emerged as a critical challenge to the implementation of targeted therapeutics. Historically, immunohistochemistry (IHC) has been used to assess spatial heterogeneity of proteins; however, it has been difficult to quantify protein abundance at high multiplex and wide dynamic range. Here, we report the development and validation of a spatially-resolved, antibody-based proteomic approach with a “barcoding-potential” to quantify up to 800 targets with 5.5 logs (base 10) of dynamic range in a single formalin-fixed paraffin-embedded (FFPE) slide. By labeling antibodies with photocleavable oligos which are recognized by NanoString® nCounter® fluorescent barcodes and subsequently exposing them to focused UV light, we have developed an nCounter assay capable of quantifying protein abundance in a predefined spatial region of a tissue section. Methods: A slide-mounted FFPE tissue section is bound with a multiplexed cocktail of primary antibody-oligo conjugates, and a microfluidic flow cell is attached to the slide. Using a simple modification of a standard microscope, regions of interest are identified by light or fluorescence microscopy and are sequentially illuminated with UV light to release the oligos. Following each illumination cycle, an eluent is collected and analyzed, resulting in digital counts that correspond to the abundance of each targeted protein in sequentially illuminated areas. We demonstrate a high degree of linearity (0.97 Application: FFPE slides from resected breast cancers are bound with an antibody cocktail (10+ plex, including HER2, EGFR, PR and others) and visualized by light microscopy. Regions of interest are identified, and oligo barcodes from those regions are released by UV illumination and digitally quantified by nCounter analysis. This enables multiplexed detection and comparison of proteins of interest from discrete regions within the tumor and adjacent normal tissue, enabling systematic interrogation of the heterogeneous tumor microenvironment. Conclusion: Application of this NanoString barcoded antibody platform to ongoing clinical studies is intended to elucidate novel responses to immunotherapy and other targeted therapies. Further development of this technology will enable the multiplexed analysis of up to 800 protein targets from a single FFPE section and facilitate detailed interrogation of spatial interactions within a tissue. The ability to measure DNA, RNA, and protein from FFPE tissue may enable the discovery of immune biomarkers in tumors and the development of companion diagnostics. Citation Format: Alessandra Cesano, Joseph Beechem, Philippa Webster, Chris Merritt, Jaemyeong Jung, Dwayne Dunaway, Gary Geiss, Sarah Warren, Gordon Mills. Spatially-resolved, multiplexed digital characterization of protein distribution and abundance in FFPE tissue sections. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1371.