Sarah Warren

Gene expression markers of Tumor Infiltrating Leukocytes

BackgroundAssays of the abundance of immune cell populations in the tumor microenvironment promise to inform immune oncology research and the choice of immunotherapy for individual patients. We propose to measure the intratumoral abundance of various immune cell populations with gene expression. In contrast to IHC and flow cytometry, gene expression assays yield high information content from a clinically practical workflow. Previous studies of gene expression in purified immune cells have reported hundreds of genes showing enrichment in a single cell type, but the utility of these genes in tumor samples is unknown. We use co-expression patterns in large tumor gene expression datasets to evaluate previously reported candidate cell type marker genes lists, eliminate numerous false positives and identify a subset of high confidence marker genes.MethodsUsing a novel statistical tool, we use co-expression patterns in 9986 samples from The Cancer Genome Atlas (TCGA) to evaluate previously reported cell type marker genes. We compare immune cell scores derived from these genes to measurements from flow cytometry and immunohistochemistry. We characterize the reproducibility of our cell scores in replicate runs of RNA extracted from FFPE tumor tissue.ResultsWe identify a list of 60 marker genes whose expression levels measure 14 immune cell populations. Cell type scores calculated from these genes are concordant with flow cytometry and IHC readings, show high reproducibility in replicate RNA samples from FFPE tissue and enable detailed analyses of the anti-tumor immune response in TCGA. In an immunotherapy dataset, they separate responders and non-responders early on therapy and provide an intricate picture of the effects of checkpoint inhibition. Most genes previously reported to be enriched in a single cell type have co-expression patterns inconsistent with cell type specificity.ConclusionsDue to their concise gene set, computational simplicity and utility in tumor samples, these cell type gene signatures may be useful in future discovery research and clinical trials to understand how tumors and therapeutic intervention shape the immune response.

Pan-cancer adaptive immune resistance as defined by the Tumor Inflammation Signature (TIS): results from The Cancer Genome Atlas (TCGA)

The Tumor Inflammation Signature (TIS) is an investigational use only (IUO) 18-gene signature that measures a pre-existing but suppressed adaptive immune response within tumors. The TIS has been shown to enrich for patients who respond to the anti-PD1 agent pembrolizumab. To explore this immune phenotype within and across tumor types, we applied the TIS algorithm to over 9000 tumor gene expression profiles downloaded from The Cancer Genome Atlas (TCGA). As expected based on prior evidence, tumors with known clinical sensitivity to anti-programmed cell death protein 1 (PD-1) blockade had higher average TIS scores. Furthermore, TIS scores were more variable within than between tumor types, and within each tumor type a subset of patients with elevated scores was identifiable although with different prevalence associated with each tumor type, the latter consistent with the observed clinical responsiveness to anti PD-1 blockade. Notably, TIS scores only minimally correlated with mutation load in most tumors and ranking tumors by median TIS score showed differing association to clinical sensitivity to PD-1/PD-1 ligand 1 (PD-L1) blockade than ranking of the same tumors by mutation load. The expression patterns of the TIS algorithm genes were conserved across tumor types yet appeared to be minimally prognostic in most cancers, consistent with the TIS score serving as a pan-cancer measurement of the inflamed tumor phenotype. Characterization of the prevalence and variability of TIS will lead to increased understanding of the immune status of untreated tumors and may lead to improved indication selection for testing immunotherapy agents.

Bringing the Next Generation of Immuno-Oncology Biomarkers to the Clinic

The recent successes in the use of immunotherapy to treat cancer have led to a multiplicity of new compounds in development. Novel clinical-grade biomarkers are needed to guide the choice of these agents to obtain the maximal likelihood of patient benefit. Predictive biomarkers for immunotherapy differ from the traditional biomarkers used for targeted therapies: the complexity of the immune response and tumour biology requires a more holistic approach than the use of a single analyte biomarker. This paper reviews novel biomarker approaches for the effective development of immune-oncology therapies, highlighting the promise of the advances in next-generation gene expression profiling that allow biologic information to be efficiently organized and interpreted for a maximum predictive value at the individual patient level.

Simultaneous, Multiplexed Detection of RNA and Protein on the NanoString ® nCounter ® Platform

The NanoString nCounter Analysis System uses a digital fluorescent barcode technology that allows for direct multiplexed measurement of gene expression (mRNA), DNA, and protein. The technology uses molecular barcodes and single-molecule imaging to detect and count unique mRNA and protein targets in a single reaction. nCounter-based detection is enzyme-free (no amplification of mRNA is required), fully automated, and allows simultaneous detection of up to 770 mRNA and 30 protein targets from multiple sample types. Target counting is fully digital with quantitative data output. Here we describe preparation of solid tumor lysate samples for use in the nCounter Analysis System.

Abstract 3814: A pan cancer analysis of the tumor inflammation signature

Introduction: The Tumor Inflammation Signature (TIS) is an 18 gene signature in development for the detection of adaptive immune response within tumors by measuring expression of genes associated with cytotoxic cells, antigen presentation, and IFNγ activity. The TIS has previously been shown to enrich for a population of patients who respond to the anti-PD1 therapy pembrolizumab. We characterized the behavior of the TIS across a range of tumor gene expression data downloaded from the TCGA in order to understand the distribution of TIS within and between tumor types, with special emphasis on those tumors for which anti-PD1 therapy is approved for use. Methods: The TIS was trained to predict response to pembrolizumab using gene expression profiles from 289 biopsies from 11 different tumor types. Its association with clinical response was retrospectively assessed in an independent set of 200 samples from 14 additional tumor types. We calculated TIS scores in over 9000 samples from 31 TCGA RNASeq datasets. We contrasted TIS with mutational load, with overall survival, and with other gene expression signatures. Results: While TIS scores are higher in classically immunogenic tumor types, they display a significant amount of intersample variability within most tumor types, and a subset of patients can be identified that possess elevated TIS scores consistent with responsiveness to pembrolizumab. TIS genes have highly conserved co-expression patterns across tumor types, consistent with a model in which the genes measure immune-intrinsic transcriptomic activity with minimal contribution of tumor-intrinsic gene expression. Notably, ranking tumors by median TIS score is superior to mutational load ranking at predicting clinical response to anti-PD1. TIS was minimally correlated with mutational load in most tumor types, except in tumor types known for hypermutation-driven by mismatch repair deficiency. TIS and mutation burden had their greatest variability in melanoma; in most other tumors, TIS retains more variability than mutation burden, possibly reflecting patients with anti-tumor immune responses driven by one or a few mutation-derived neoantigens. Average mutation burden and average TIS are positively correlated within most tumor types. Notable exceptions to this trend include two tumors for which anti-PD1 is currently approved. Bladder urothelial carcinoma has lower average TIS than its mutation burden would predict, and kidney renal clear cell carcinoma has much higher average TIS than its mutation burden would predict. Conclusion: The TIS has the potential to enrich for anti-PD1 responders independent of tumor type. It may identify rare potential responders in tumors with low average response rates. These results support further research into the efficacy of anti-PD1 in patients with high TIS scores, regardless of tumor type. Citation Format: Patrick Danaher, Sarah Warren, Alessandra Cesano. A pan cancer analysis of the tumor inflammation signature [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 3814. doi:10.1158/1538-7445.AM2017-3814

Abstract PD5-06: Prognostic value of molecular tumor infiltrating lymphocyte (mTIL) signatures in HER2-positive breast cancer patients in N9831 and FinHer/FinXX trials

Background : While previous study showed that the enrichment of immune-related gene expression was associated with outcome in HER2+ patients receiving sequential or concurrent trastuzumab (H), stromal tumor infiltrating lymphocytes (sTIL) have not been consistently shown to associate with outcome in this group of patients. Given that TIL scoring may be subjective, we analyzed molecular signatures of different subsets of tumor infiltrating immune cell populations, using NanoString TM gene expression data to assess molecular TIL (mTIL) signature enrichment and intrinsic subtype as a function of relapse-free survival (RFS). Methods : NanoString TM technology was used to quantify mRNA in samples from 1,280 patients in N9831, 168 patients in FinHer, and 170 patients in FinXX. In N9831, patients in arm A were treated with chemotherapy alone (AC-T), arm B received chemotherapy followed by sequential H (AC-T-H), and arm C received H concurrently with chemotherapy (AC-TH). In the FinHer trial, H was given concurrently for 9 weeks and either 1 year or 9 weeks in FinXX trial. Cox proportional hazard ratio (HR) was used to determine the association of each gene signature with RFS. Different immune subset signatures, including CD45, B-cells, CD8 T-cells, cytotoxic-cells, and T-cells were analyzed using algorithms developed by NanoString. Results : In N9831, CD45, cytotoxic-cell, and T-cell signatures were significantly associated with improved RFS in patients receiving chemotherapy alone and AC-T-H. However, none of the mTIL signatures were significantly associated with outcome in patients receiving AC-TH. Patients lacking CD45 enrichment had better outcome when H was given concurrently with chemotherapy. The 10-year Kaplan-Meier estimates for RFS in arm B patients with CD45 enrichment or no enrichment were 81.3% and 72.6%, respectively (HR 0.63 [95% CI, 0.42-0.93]; p = 0.02), and in arm C were 83.6% and 79.8%, respectively (HR 0.79, 95%CI 0.49-1.28; p = 0.34). Among patients with HER2-enriched subtype, all of the mTIL signatures were associated with improved RFS in arm A (AC-T) and B (AC-T-H) but remained non-significant in arm C (AC-TH). In patients with luminal subtypes, mTIL signatures were not significantly associated with outcome in patients treated with chemotherapy alone. Similar findings were observed in the FinHer and FinXX trials, in which, none of mTIL signatures were significantly associated with outcome among patients who received H. Conclusion : This analysis sheds light on previous discrepancy between immune-related gene signature and sTIL findings. Our data also suggests that the poor prognosis associated with lack of infiltrating immune cells can be partly overcome by the concomitant administration of H with chemotherapy. mTIL signatures, specifically CD45, cytoxic, and T cells, were prognostically associated with improved outcome in patients receiving chemotherapy without concurrent trastuzumab. Understanding the role of the immune system in response to H will require a higher degree of granularity than can be achieved by histological quantification of TILs. Further studies are needed to validate the significance of mTIL signatures as predictive or prognostic biomarker in HER+ patients. Citation Format: Chumsri S, Serie DJ, Mashadi-Hossein A, Tenner KS, Lauttia SL, Moreno-Aspitia A, McLaughlin SA, Nassar A, Warren S, Danaher P, Colon-Otero G, Lindman H, Joensuu H, Perez EA, Thompson EA. Prognostic value of molecular tumor infiltrating lymphocyte (mTIL) signatures in HER2-positive breast cancer patients in N9831 and FinHer/FinXX trials [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr PD5-06.

Placental immune editing switch (PIES): learning about immunomodulatory pathways from a unique case report

The hypothesis of this work is that, in order to escape the natural immune surveillance mechanisms, cancer cells and the surrounding microenvironment might express ectopically genes that are physiologically present in the placenta to mediate fetal immune-tolerance. These natural “placental immune-editing switch” mechanisms (PIES) may represent the result of millions of years of mammalian evolution developed to allow materno-fetal tolerance. Here, we introduce genes of the immune regulatory pathways that are either similarly over- or under-expressed in tumor vs normal tissue. Our analysis was carried out in primary breast cancer with metastatic homolateral axillary lymph nodes as well as placenta tissue (both uterine decidual tissue and term placenta tissue) from a pregnant woman. Gene expression profiling of paired non-self and self tissues (i.e. placenta/uterus; breast cancer/normal breast tissue; metastatic lymphnode/normal lymphnode tissue) was performed using the PanCancer Immune gene panel, a 770 Nanostring gene expression panel. Our findings reveal overlapping in specific immune gene expression in placenta and cancer tissue, suggesting that these genes might play an important role in maintaining immune tolerance both physiologically (in the placenta) and pathologically (in the cancer setting).

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 B095: Multiplexed detection of RNA and proteins on the nCounter® platform with low sample input protocol

As our understanding of the immune responses to cancer continues to grow, the need to extract greater amounts of information from ever smaller sample sizes increases. One of the biggest challenges facing the field is to develop a comprehensive understanding of how the immune system responds to a tumor, and multi-omic profiling (DNA, RNA, and protein) is crucial to building a holistic model of tumor immunity. The NanoString nCounter platform has become an important tool in quantifying transcriptional responses from a wide variety of sample types by enabling direct digital counting of up to 800 targets from a single sample using nucleic acid probes that directly hybridize to the RNA sequence of interest and then are quantified with optical barcoding technology. The platform has now been extended to permit quantification of proteins at the same time using primary antibodies conjugated to DNA oligos that hybridize with the barcodes. We have developed new technology that enables characterization of up to 30 proteins and 770 RNA transcripts in key immuno-oncology pathways from a very low amount of starting material - as few as 50,000 cells. NanoString has previously developed the nCounter Vantage RNA:Protein Immune Profiling Panel which allows digital counting of extracellular proteins which facilitates quantitation of multiple immune cell populations and provides information about their activation status. We have now applied this technology to detect intracellular and secreted proteins as well in the new nCounter Vantage RNA:Protein Immune Signaling panel. This panel is able to detect key transcription factors, signaling molecules, and secreted proteins from peripheral blood mononuclear cells (PBMC), dissociated cells, or cell culture. Additionally, we have recently developed a universal cell capture technology that utilizes anti-β2M antibody coupled to magnetic beads to pull down nucleated cells from a large starting volume. As proof of concept, PBMC from a healthy donor were treated with phorbol 12-myristate 13-acetate (PMA) and ionomycin, TNFα, or IFNγ and RNA and protein were characterized with both the Immune Profiling and Immune Signaling panels. nCounter protein detection compared favorably when validated with flow cytometry, and the additional information imparted by simultaneous profiling of the RNA transcriptome enabled mapping of signaling pathways activated by treatment. Furthermore, RNA and protein counts from the low input protocol were representative of counts obtained from higher cell inputs, indicating that no loss or skewing of data resulted from reducing the starting material. This advance in multi-analyte, multiplexed digital molecular profiling will accelerate immuno-oncology research by reducing sample size requirements and may enable the discovery and development of novel immunotherapies and their associated companion diagnostics. Citation Format: Sarah Warren, Gary Geiss, Brian Burditt, Qian Mei, Alan Huang, Maribeth Eagen, Eduardo Ignacio, Dwayne Dunaway, Lucas Dennis, Joseph Beechem. Multiplexed detection of RNA and proteins on the nCounter® platform with low sample input protocol [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 B095.

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.