Philippa Webster

Direct multiplexed measurement of gene expression with color-coded probe pairs

We describe a technology, the NanoString nCounter gene expression system, which captures and counts individual mRNA transcripts. Advantages over existing platforms include direct measurement of mRNA expression levels without enzymatic reactions or bias, sensitivity coupled with high multiplex capability, and digital readout. Experiments performed on 509 human genes yielded a replicate correlation coefficient of 0.999, a detection limit between 0.1 fM and 0.5 fM, and a linear dynamic range of over 500-fold. Comparison of the NanoString nCounter gene expression system with microarrays and TaqMan PCR demonstrated that the nCounter system is more sensitive than microarrays and similar in sensitivity to real-time PCR. Finally, a comparison of transcript levels for 21 genes across seven samples measured by the nCounter system and SYBR Green real-time PCR demonstrated similar patterns of gene expression at all transcript levels.

Modeling Bi-modality Improves Characterization of Cell Cycle on Gene Expression in Single Cells

Advances in high-throughput, single cell gene expression are allowing interrogation of cell heterogeneity. However, there is concern that the cell cycle phase of a cell might bias characterizations of gene expression at the single-cell level. We assess the effect of cell cycle phase on gene expression in single cells by measuring 333 genes in 930 cells across three phases and three cell lines. We determine each cells phase non-invasively without chemical arrest and use it as a covariate in tests of differential expression. We observe bi-modal gene expression, a previously-described phenomenon, wherein the expression of otherwise abundant genes is either strongly positive, or undetectable within individual cells. This bi-modality is likely both biologically and technically driven. Irrespective of its source, we show that it should be modeled to draw accurate inferences from single cell expression experiments. To this end, we propose a semi-continuous modeling framework based on the generalized linear model, and use it to characterize genes with consistent cell cycle effects across three cell lines. Our new computational framework improves the detection of previously characterized cell-cycle genes compared to approaches that do not account for the bi-modality of single-cell data. We use our semi-continuous modelling framework to estimate single cell gene co-expression networks. These networks suggest that in addition to having phase-dependent shifts in expression (when averaged over many cells), some, but not all, canonical cell cycle genes tend to be co-expressed in groups in single cells. We estimate the amount of single cell expression variability attributable to the cell cycle. We find that the cell cycle explains only 5%–17% of expression variability, suggesting that the cell cycle will not tend to be a large nuisance factor in analysis of the single cell transcriptome.

Continuously tunable nucleic acid hybridization probes

In silico–designed nucleic acid probes and primers often do not achieve favorable specificity and sensitivity tradeoffs on the first try, and iterative empirical sequence-based optimization is needed, particularly in multiplexed assays. We present a novel, on-the-fly method of tuning probe affinity and selectivity by adjusting the stoichiometry of auxiliary species, which allows for independent and decoupled adjustment of the hybridization yield for different probes in multiplexed assays. Using this method, we achieved near-continuous tuning of probe effective free energy. To demonstrate our approach, we enforced uniform capture efficiency of 31 DNA molecules (GC content, 0–100%), maximized the signal difference for 11 pairs of single-nucleotide variants and performed tunable hybrid capture of mRNA from total RNA. Using the Nanostring nCounter platform, we applied stoichiometric tuning to simultaneously adjust yields for a 24-plex assay, and we show multiplexed quantitation of RNA sequences and variants from formalin-fixed, paraffin-embedded samples.

Digital multiplexed mRNA analysis of functionally important genes in single human oocytes and correlation of changes in transcript levels with oocyte protein expression

Objective To investigate functionally important transcripts in single human oocytes with the use of NanoString technology and determine whether observed differences are biologically meaningful. Design Analysis of human oocytes with the use of NanoString and immunoblotting. Setting University-affiliated reproductive medicine unit. Patients Women undergoing in vitro fertilization. Intervention Human oocytes were analyzed with the use of NanoString or immunoblotting. Main Outcome Measures The abundance of transcripts for ten functionally important genes—AURKA, AURKC, BUB1, BUB1B (encoding BubR1), CDK1, CHEK1, FYN, MOS, MAP2K1, and WEE2—and six functionally dispensable genes were analyzed with the use of NanoString. BubR1 protein levels in oocytes from younger and older women were compared with the use of immunoblotting. Result(s) All ten functional genes but none of the six dispensable genes were detectable with the use of NanoString in single oocytes. There was 3- to 5-fold variation in BUB1, BUB1B, and CDK1 transcript abundance among individual oocytes from a single patient. Transcripts for these three genes—all players within the spindle assembly checkpoint surveillance mechanism for preventing aneuploidy—were reduced in the same oocyte from an older patient. Mean BUB1B transcripts were reduced by 1.5-fold with aging and associated with marked reductions in BubR1 protein levels. Conclusion(s) The abundance of functionally important transcripts exhibit marked oocyte-to-oocyte heterogeneity to a degree that is sufficient to affect protein expression. Observed variations in transcript abundance are therefore likely to be biologically meaningful, especially if multiple genes within the same pathway are simultaneously affected.

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 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.

Abstract 5100: Development of a direct-measurement molecular assay to determine telomere length in human samples

Telomere length (TL) is a recognized biomarker in cancer. Epidemiology studies suggest that blood and/or buccal cell TL is associated with certain cancers; however, TL measurement is still an under-utilized biomarker in clinical trials. The molecular testing platforms currently used for TL determination are not optimal due to inherent technical limitations. While qPCR based assays have high throughput and are inexpensive, they are difficult to develop and are very sensitive to subtle permutations in reaction components. We evaluated a strategy that relies on direct hybridization and detection of the telomere target utilizing the NanoString nCounter System (NS). The NS is a semi-quantitative assay that compares the number of reporter probe hybridization events of telomere-specific versus chromosomal invariant regions in DNA samples. Performed without nucleic acid amplification, it has potential to more accurately quantify telomeric sequences than any other assay currently in use. NS uses moderate DNA quantities per test (600ng), and has a throughput that can be expanded in multiples of 12. In each sample, the Ratio of Telomere Length (RTL) is obtained by normalizing the signal produced by the telomere specific code set probes to the signal produced by multiple control probes as compared to commercial normal human DNA. NS was validated in a three stage process using commercial DNA (n=3), cell lines (n=11) and human clinical samples (n=43) that had previously been tested by other TL tests. A blinded panel of six research samples previously tested in-house by multiplex monochrome quantitative PCR (qPCR) was sent to NanoString for in-house testing. NS exactly reproduced the panel9s previous RTL ranking. The telomere code sets were subsequently tested at NCI on a panel of 11 established cell lines. Good correlation was observed compared with our in-house qPCR assay (R2 = 0.89). For the third stage of the validation, we evaluated the assay9s utility on human clinical samples for which 43 whole blood leukocyte DNA samples were tested (18 dyskeratosis congenita (DC) patients with very short TL, 25 mutation-free (normal) DC relatives) and compared against leukocyte flow cytometry with in situ hybridization (flow-FISH, FF) TL measurement; FF is the gold standard in clinical testing for DC. The correlation between NS and FF lymphocyte TL was high, R2 = 0.72. Notably, the two sample t test showed that the NS assay could discriminate RTL between normals and DC patients (p = 4.6x10-8); normal relatives have longer telomeres. These data suggest that the NS RTL assay has the ability to be specific and sensitive to differences in TL between individuals with DC and healthy individuals. To determine the assay9s exact clinical parameters a greater number of samples will need to be tested. The NS assay is less costly than the FF test by at least one half and if shown to be reproducible would be a viable alternative to FF and qPCR, especially in a diagnostic setting. Citation Format: Daniel C. Edelman, David Petersen, Allison Gomez, Shahinaz Gadalla, Philippa Webster, Lucas Dennis, Mike Krouse, Sharon Savage, Paul S. Meltzer. Development of a direct-measurement molecular assay to determine telomere length in human samples. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5100. doi:10.1158/1538-7445.AM2014-5100