Gene Expression Panel Research Articles

Abstract 5963: NGS-based murine pan-cancer gene expression panel for immuno-oncology and tumor microenvironment studies

Abstract INTRODUCTION: Cancer is a heterogeneous disease and highly related to the immunological system. The significance of fully understanding the interactions between cancer and immunity in cancer therapeutics has been broadly noted, particularly for immunotherapies. Experimental murine models derived from a variety of biological technologies are essential preclinical model systems utilized before clinical studies. Molecular pathology techniques, such as gene expression biomarkers, are widely used to systematically profile and characterize these models. So far, however, there has been no robust and cost-effective assay designed for murine clinical immuno-oncology (I/O) research to characterize tumor-immune interactions at a gene expression level. METHODS: Marker genes were identified by combining public and in-house reference genomic data for non-tumor bearing mice as well as tumor mouse models. Oligonucleotide-based hybridization/capture techniques analogous to exome sequencing, targeting specific regions are well established on Illumina Novaseq and BGI MGIseq platforms. These methods were applied to the murine I/O panel assay, with accuracy and reproducibility verified across these two sequencing platforms. The results were further qualified using a PCR array platform. RESULTS: A mouse gene panel involving 1080 genes that encompass surface markers and transcriptomic biomarkers for the immune system, key pathways at the interface of the tumor, tumor microenvironment (TME), and immune response, as well as internal reference genes for data normalization has been established. By combining advanced NGS technologies and specific bioinformatic pipelines, we were able to detect and distinguish various immune and stromal cells involved in the complex interaction between the tumor and its microenvironment, allowing for a multifaceted characterization of disease biology and the interrogation of immune response and evasion. We validated the sensitivity, specificity, and repeatability of the panel by qPCR experiments. CONCLUSIONS: We have established a murine-I/O NGS panel involving 1080 genes to characterize tumor-immune interactions more efficiently and in a cost-effective manner for preclinical studies. This provides an enhanced insight into the TME, with high-content data that has the potential to translate more effectively to the clinic as well as for early identification of immunotherapy companion diagnostics. Citation Format: Xiaobo Chen, Jia Xue, Qixiang Li, Sheng Guo. NGS-based murine pan-cancer gene expression panel for immuno-oncology and tumor microenvironment studies [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 5963.

Abstract LB-351: Investigating the role immunogenomics in normal adjacent to tumor tissue (NAT) versus tumor in prognostication of stage matched colorectal cancer

Abstract Colorectal cancer (CRC) is the third most common type of cancer affecting nearly 1.4 million individuals every year. In the past two decades, rapid advancements in genomics and transcriptomics have uncovered various molecular profiling methods that are assisting in the design of personalized treatment strategies for cancer patients. In this study, we attempted to identify differentially expressed genes in tumor and NAT for its clinical significance in CRC. We designed a novel 88 gene-expression panel consisting of immune and cancer driver genes based on literature. Under IRB approved protocol, 28 FFPE samples (tumor = 14, NAT = 14) were acquired from pathology archives. H&E slides were examined and by using macrodissection, regions with >95% of tumor or NAT region were identified by two expert pathologist. Total RNA was isolated from these tissue sections and quantified through Nanodrop method. The expression level of the genes was measured using NanoString digital quantification system. Pairwise comparisons by Wilcoxon rank-sum test identified significant gene perturbations in the NAT compared to the tumor. IL-15, GATA3, CCR4, CCL13 and IFNA1 genes were highly expressed in NAT (p <0.05). Comparatively, PKM, E2F1, GTF3A, MCM4, MMP14 genes were expressed at a higher amount in tumor compared to NAT (p <0.05). Unsupervised hierarchical clustering identified two distinct clusters belonging to tumor and NAT. String DB analysis further identified immune genes as a dominant node with higher expression in NAT. Comparatively, tumors showed higher expression of genes belonging to the cell growth pathway. This observation identifies the predominance of “cold” immune status of tumors compared to immunologically active adjoining regions. Interestingly, the expression of IKBKB (Inhibitor of NFKB Kinase Subunit Beta) was found to be lower in patients that received chemotherapy. IKBKB helps in the activation of NFKB complex by phosphorylation and dissociation of the inhibitor of NFKB. This study has identified immunologically active tumor adjacent normal regions and the variable expression of IKBKB in patients who received chemotherapy. The clinical significance of these findings will be further explored in large patient cohorts. Further, there is a need to comprehensively explore more functionally relevant genes in NAT regions that can have prognostic and predictive potential. The expanded understanding of differential gene expression of NAT compared to the tumor might assist in the design of novel combinatorial therapies. Citation Format: Pankaj Kumar Ahluwalia, Ashis Mondal, Meenakshi Ahluwalia, Nikhil Sahajpal, Allan Njau, Nwogbo Okechukwu, Vamsi Kota, Ravindra Kolhe. Investigating the role immunogenomics in normal adjacent to tumor tissue (NAT) versus tumor in prognostication of stage matched colorectal cancer [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 LB-351.

Abstract A52: Targeted expression profiling identifies mechanisms of potential breast carcinogens

Abstract Over 200 chemicals have been associated with altered mammary gland development and cancer in rodent studies, yet the molecular mechanisms by which this diverse set of chemicals affect the breast remain poorly understood. We examined gene expression profiles of eight known and suspected mammary toxicants using the TempO-Seq platform and a targeted gene expression panel, Breast Carcinogen Screen (BCScreen). The BCScreen panel has 500 genes with at least 30 genes strongly associated with each of the 14 biologic processes relevant to breast cancer and mammary gland development. We compared gene expression profiles in MCF-7 cells treated for 24hrs with four concentrations (1 nM to 10 uM) of the following chemicals: three estrogenic chemicals (genistein, bisphenol A, butyl benzyl phthalate); the selective estrogen receptor modulator, tamoxifen (TAM); 1,4 benzoquinone (BQ), a genotoxic metabolite of the rodent mammary carcinogen benzene; perfluorooctanoic acid (PFOA), a persistent pollutant that alters mammary gland development; and two closely related perfluorinated chemicals that have not been evaluated for mammary toxicity/effects (PFHXA, PFNA). We identified expression patterns activated by mammary toxicants with known ER agonist activity (genistein, BPA, BBP). Some of these genes were suppressed by PFOA and BQ and TAM, mammary toxicants with antagonist or no known direct ER activity. The known weak estrogens genistein, BPA, and BBP had similar gene expression profiles and were enriched for genes associated with cell cycle and DNA repair, particularly at higher doses. Notably, PFOA, Tam, and BQ had greater gene expression changes at lowest (1nM) vs. higher doses and were enriched for genes associated with cell cycle. For context, serum PFOA levels in the general US population are generally five to ten times higher than the 1 nM dose where we observed these changes (i.e., > 0.4 micrograms/L). PFOA profiles were enriched for several transcription factors including E2F1 and E2F4, which regulate cell cycle genes and important processes in mammary development and tumor progression. Utilizing the BCScreen gene panel, we have identified mechanistic targets relevant to breast cancer that are perturbed by mammary toxicants. Citation Format: Vanessa Y. De La Rosa, Erik Lehnert, Marko Milanovic, Chris Vulpe, Ruthann A. Rudel. Targeted expression profiling identifies mechanisms of potential breast carcinogens [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A52.

Abstract A01: HPV+ oral cancer PDXs identify prognostic utility for E2F target gene expression and mutation burden

Abstract Barriers to advancing therapy for HPV-related (HPV+) head and neck squamous cell carcinomas (HNSCCs) include a paucity of preclinical models and difficulty prospectively identifying the minority of cases with lethal potential. This study therefore sought to establish HPV+ patient-derived models with high genetic fidelity and use them to help identify aggressive molecular subtypes of HPV+ HNSCC. Using whole-exome sequence and viral transcript analysis, nine stably passaged patient-derived xenogafts (PDXs) were found to avoid typical artifacts in HPV+ cell lines, which include loss of canonical PIK3CA mutants, gains in 3q copy number, and increased viral integration. Notably, the models contained markedly increased rates of alteration in Notch pathway genes and in H3K4 methyltransferases than would be predicted by The Cancer Genome Atlas (TCGA). Although phenotypic correlates of these alterations were not overtly apparent in the models, analysis of their growth features uncovered strong positive associations of tumor mutational burden (TMB) with in vivo growth rate and in vitro organoid propagation capacity. This observation led to identification of a novel association in TCGA between TMB and both reduced local progression and survival of early HPV-negative HNSCCs, which was further validated in esophageal squamous cell carcinomas. Although TMB failed to be prognostic in the HPV+ HNSCCs, insight into high-risk HPV+ molecular features was provided by a PDX from an unusual case that suffered lethal relapse shortly after definitive therapy. Reduced viral E7 and p16INK4A expression was identified both in this model and in early lethal HPV+ cases in TCGA as well as those in a second HPV+ HNSCC cohort (JHU). The lower E7 and p16INK4A levels here suggested a diminished imprint of viral oncogene function upon cell cycle regulation in lethal cases. Hierarchical clustering of the cohorts using a broad E2F target gene expression panel revealed a distinctive pattern of E2F target dysregulation in the clusters that captured the early lethal cases. A subset of ten transcripts optimally predicted survival independent of stage in both TCGA and JHU cohorts. This analysis of the largest panel of HPV+ HNSCC PDXs described to date identified robust models for PIK3CA activation, Notch pathway dysregulation, and altered H3K4 methylation in this disease. At the same time, it defined novel clinical correlates of TMB and E2F target expression pattern that have potential utility in guiding risk stratification, biomarker development, and trial design. Citation Format: Nicole D. Facompre, Pavithra Rajagopalan, Alexander T. Pearson, Kathleen T. Montone, Claire D. James, Frederico O. Gleber-Netto, Hiroshi Nakagawa, Elizabeth A. White, Anil K. Rustgi, Joseph A. Califano, Curtis R. Pickering, Bradley Windle, Iaian M. Morgan, Phyllis A. Gimotty, Devraj Basu. HPV+ oral cancer PDXs identify prognostic utility for E2F target gene expression and mutation burden [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr A01.

Abstract B40: Analytical validation and initial clinical utility of multianalyte transcriptomic biomarker profiling of circulating tumor cells using automated exclusion-based sample preparation technology

Abstract The efficacy of androgen receptor (AR) signaling inhibitors (ARSIs) in prostate cancer is limited by both de novo and acquired resistance. Two well-described mechanisms of resistance to ARSIs are the development of constitutively activated AR splice variants (AR-Vs) or neuroendocrine prostate cancer (NEPC). Expression of NEPC markers is traditionally evaluated in solid tumor biopsies while AR-Vs can be detected in circulating tumor cells (CTCs) and have prognostic relevance in prostate cancer. Unlike solid tumor biopsy-based detection strategies, liquid biopsies can be performed periodically over the course of treatments, enabling early identification of resistance mechanisms. To evaluate for ARSI resistance, we developed a multiplex gene expression panel of AR-Vs and NEPC markers. We evaluated potential clinical utility of this assay in CTCs captured using an exclusion-based sample preparation technology. This test was evaluated with a cohort of 46 patients, showing 39% sensitivity and 94% specificity at detecting ARSI resistance. To develop a clinical-grade liquid biopsy assay with this multiplexed gene expression panel, we utilized a high-throughput automated exclusion-based sample preparation (ESP) technology for CTC capture, mRNA extraction, and gene expression analysis using RT-qPCR. Cell lines were used to evaluate the analytical performance of the methodology, and spike-in control materials were designed to monitor the efficiency of mRNA extraction for each individual patient sample. Serial dilutions of cell lines demonstrated the sensitivity to extract and quantify mRNA at the single-cell level with high precision between evaluations performed on three different days. Increasing numbers of cells resulted in the quantification of proportionally higher amounts of both AR-Vs and NEPC markers, demonstrating the analytical accuracy of the methodology (R2 values of 0.8 and above). To monitor for RNA degradation, a grape mRNA amplicon material was designed as an internal spike-in control for daily use in testing patient samples. Healthy donor PBMCs were used to confirm primers were specific for cells of cancer origin. ESP-mRNA extraction followed by RT-qPCR demonstrates single-cell resolution for the detection of AR-V/NEPC transcriptomic expression, with high precision in evaluating actual patient samples. The high analytical performance, and the promising preliminary correlation with ARSI resistance, warrant further efforts towards implementation into clinical laboratory testing environments and testing in prospective clinical trials. Citation Format: Zachery D. Schultz, Jennifer L. Schehr, Rory M. Bade, Molly M. Morgan, Madalyn S. Gill, Hannah M. Pezzi, Jaime M. Sperger, Charlotte N. Stahlfeld, Anupama Singh, Jay W. Warrick, David J. Beebe, Joshua M. Lang. Analytical validation and initial clinical utility of multianalyte transcriptomic biomarker profiling of circulating tumor cells using automated exclusion-based sample preparation technology [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B40.

Sa1466 EUS HAS THE POTENTIAL TO EVALUATE MEMBERS OF THE TUMOR NECROSIS FACTOR (TNF) SUPERFAMILY AND IDENTIFY DRUG TARGETS ON AN INDIVIDUAL PDAC PATIENT BASIS TO GUIDE PRECISION IMMUNE-ONCOLOGY ELIGIBILITY

The FDA has approved use of the programmed cell death-1 (PD-1) inhibitor, pembrolizumab, in MSI-High solid tumors. This is a rare event among pancreas ductal adenocarcinoma (PDAC) patients, occurring at a frequency of 0.8%. In order to decipher if other cancer immunotherapy targets are potential therapies for PDAC patients, the Tumor Necrosis Factor (TNF) Receptor Superfamily has gained prominence, due to its ability to activate antitumor T-cell immunity. Members include CD70, TNFSF4 (OX-40L) and TNFRSF9 (41BB). It is important to develop strategies to aid patient selection criteria to guide therapeutic decision-making for clinical trials. We sought to determine if EUS fine needle biopsy (FNB) treatment naïve PDAC specimens are suitable to evaluate the TNF Superfamily, and in turn identify potential targets on an individual patient basis. The cohort of 13 EUS FNB patient specimens, 7 of whom with ≥ stage III disease, a CA19-9 level of 227.5 (IQR 58-605) U/ml and overall mortality of 77% at 22.2 (IQR 13.4-36.1) months was evaluated by the Nanostring nCounter® PanCancer Immune Profiling Panel. This is a highly multiplexed mRNA gene expression panel designed to quantitate the expression of 770 genes. A ≥ 2-fold change in expression when compared to pancreas control tissue was deemed to be reflective of altered expression. The digital mRNA profiling panel revealed that 44.8% and 10% of evaluated genes had either increased or reduced expression, when compared to normal pancreas controls. Of the 30 TNF Superfamily gene members evaluated, 17 (56.7%) and 1 (3.3%) were up and downregulated in PDAC and 4 (13.3%) were each up and down regulated in surgical resection chronic pancreatitis specimens (n=3), respectively. (Figure 1) TNFRSF10C (alias TRAILR3) and TNFRSF11B (alias Osteoprotegerin) expressions were elevated in 12 (92.3%) PDAC specimens x12.2-fold (range x1.8 – x36.2 fold) and x6.5-fold (range x1.8 – x40.9 fold), respectively. CD70, an immune checkpoint, had a tumor-restricted expression pattern and was upregulated in 10 (77%) PDAC patients (x3.2-fold) but downregulated in chronic pancreatitis (x-4.23-fold). (Figure 2) EUS has a central role in guiding precision immune-oncology eligibility for clinical care and clinical trials. Using digital mRNA profiling, CD70 had a tumor-restricted expression pattern and was identified in 77% of PDAC patient specimens. Therefore, this suggests that such patients may be sensitive to monoclonal antibody therapy that targets and neutralizes CD70.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Abstract A111: Urine-based gene expression panels with focus on prostate cancer in African Americans

Abstract Introduction: Prostate cancer (CaP) affects 1 in 7 men in their lifetime. African American (AA) men have significantly higher incidence and mortality from CaP compared to Caucasian American (CA) men. We and others have also noted that the genes commonly overexpressed in CaP (e.g., ERG) and currently used as diagnostic markers exhibit much lower frequency and more heterogeneity in AA men. The goals of this study are to develop CaP diagnostic/prognostic marker panels that takes into account race-associated differences in molecular alterations. Here we evaluated a panel of CaP associated genes using regular (non-DRE) urine from AA and CA CaP for discriminating between high-grade (Gleason score [GS], 7-10) and low-grade disease (GS, 6) and cancer negative biopsy. Methods: The quantitative expression assay protocols have been developed for noninvasive detection of candidate genes in exosomal RNAs from regular urine. The selected genes were reverse transcribed in a single reaction using gene-specific primer pool (GSP) followed by preamplification and PCR-based assay of all target genes. Results: Regular urine exosomal RNA-based assays were developed and optimized for PSGR, DLX1, HOXC4, NKX2-3, COL10A1, HOXC6, PCA3, PCGEM1, PCAT1, CTBP1, SChLAP1, ERG and SPDEF. All markers have been evaluated in a pilot study of 72 subjects undergoing diagnostic biopsies. For AA patients a sensitivity of 88.2%, specificity of 83.3%, and AUC of 0.81 were achieved for the panel (PCA3, PCGEM1, HOXC6 and SChLAP1). Consistent with numerous studies, ERG and PCA3 panel was optimal for CA patients. Preliminary analysis showed that 3 markers were found to be associated with improved discrimination between GS7 or greater and GS6 (AUC 0.78 for ERG; AUC 0.72 for PSGR and AUC 0.72 for SChLaP1) in AA CaP. In CA patients, PCA3 gave an AUC of 0.76 for predicting high-grade from low-grade disease. Analysis of an additional 100 patients is in progress. Conclusions: Given the genomic difference of prostate cancer among different race/ethnicity, this study highlights the potential for developing CaP biomarker panels in the context of race for enhancing precision medicine through improved CaP diagnosis/prognosis Citation Format: Indu Kohaar, Sreedatta Banerjee, Yongmei Chen, Amina Ali, Jacob Kagan, Sudhir Srivastava, Jennifer Cullen, Inger Rosner, Shiv Srivastava, Gyorgy Petrovics. Urine-based gene expression panels with focus on prostate cancer in African Americans [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr A111.

Platform comparison of HTG EdgeSeq and RNA-Seq for gene expression profiling of tumor tissue specimens.

3566 Background: Clinical biomarker studies are often hindered by the availability of tissue specimens of sufficient quality and quantity. While RNA-Seq is often considered the gold standard for measuring mRNA expression levels in cancer tissue, it typically requires multiple formalin-fixed paraffin-embedded (FFPE) tissue sections to extract a sufficient amount of quality RNA for subsequent gene expression profiling analysis. The HTG EdgeSeq technology is a gene expression profiling platform that combines quantitative nuclease protection assay technology with next-generation sequencing detection. Unlike RNA-Seq, the HTG EdgeSeq technology does not require RNA extraction, and can use small amounts of tissue material, typically several mm2, to generate reproducible gene expression profiles. Methods: This study compares the performance of RNA-Seq and HTG's profiling panel, the HTG EdgeSeq Precision Immuno-Oncology Panel (PIP), which is designed to measure expression levels of 1,392 genes focused on tumor/immune interaction. Approximately 1,200 samples from three tumor indications (gastric cancer, colorectal cancer and ovarian cancer) were tested using both technologies. Results: Up to four FFPE slides were used for RNA extraction to support RNA-Seq testing; out of the 1,202 samples processed, 1,099 generated extracted RNA of sufficient quality and quantity (as measured by RNA concentration, RIN score and %DV200) to proceed to sequencing, which resulted in a pass rate of 91.4% for RNA-Seq. The HTG EdgeSeq PIP panel resulted in a pass rate of 97.3% (samples passing QC metrics) when the same 1,200 samples were tested, and required only a single FFPE section owing to the small sample requirement. The t-SNE (a non-linear dimensionality reduction method) analysis of the common 1,358 genes revealed similar clustering of the three cancer indications between the two methods. Correlations across individual genes by sample resulted in the mean Spearman correlation coefficient of 0.73 (95% confidence interval of 0.61 - 0.80). Additionally, gene-wise comparisons across all samples were also evaluated. Conclusions: These data demonstrate that HTG EdgeSeq gene expression panels can be used as a competitive alternative to RNA-Seq, generating equivalent gene expression results, while offering the added benefits of a small sample size requirement, lack of RNA extraction bias, and fully automated data analysis pipeline.

Evolving Landscape of Molecular Prescreening Strategies for Oncology Early Clinical Trials.

Most academic precision oncology programs have been designed to facilitate enrollment of patients in early clinical trials with matched targeted agents. Over the last decade, major changes were seen both in the targetable molecular alteration landscape and in drug development trends. In this article, we describe how the Vall d’Hebron Institute of Oncology molecular prescreening program adapted to a dynamic model of biomarker-drug codevelopment. We started with a tumor-agnostic hotspot mutation panel plus in situ hybridization and immunohistochemistry of selected markers and subsequently transitioned to tumor-specific amplicon-based next-generation sequencing (NGS) tests together with custom copy number, fusion, and outlier gene expression panels. All assays are optimized for archived formalin-fixed paraffin-embedded tumor tissues without matched germline sequencing. In parallel, biomarker-matched trials evolved from a scenario of few targets and large populations (such as PI3K inhibitors in PIK3CA mutants) to a complex situation with many targets and small populations (such as multiple targetable fusion events). Recruitment rates in clinical trials with mandatory biomarkers decreased over the last 3 years. Molecular tumor board meetings proved critical to guide oncologists on emerging biomarkers for clinical testing and interpretation of NGS results. The substantial increase of immunotherapy trials had a major impact in target prioritization and guided clinical implementation of new markers, such as tumor mutational burden, with larger exon-based NGS assays and gene expression signatures to capture microenvironment infiltration patterns. This new multiomics era of precision oncology is expected to increase the opportunities for early clinical trial matching.

1161 CLINICAL AND HOST BIOLOGICAL FACTORS CAN PREDICT COLECTOMY RISK IN CHILDREN NEWLY DIAGNOSED WITH ULCERATIVE COLITIS

BACKGROUND Develop a clinical and biological predictive model for colectomy risk in children newly diagnosed with ulcerative colitis (UC). METHODS This was a multicenter inception cohort study of children (ages 4-17 years) newly diagnosed with UC treated with standardized initial regimens of mesalamine or corticosteroids (CS) depending upon initial disease severity. Therapy escalation to immunomodulators or infliximab was based on predetermined criteria. Patients were phenotyped by clinical activity per the Pediatric Ulcerative Colitis Activity Index (PUCAI), disease extent, endoscopic/histologic severity, and laboratory markers. In addition, RNA sequencing defined pretreatment rectal gene expression and high density DNA genotyping by the Affymetrix UK Biobank Axiom Array. Coprimary outcomes were colectomy over 3 years and time to colectomy. Generalized linear models, Cox proportional hazards multivariate regression modeling, and Kaplan-Meier plots were used. RESULTS Four hundred twenty-eight patients (mean age 13 years) started initial theapy with mesalamine (n = 136), oral CS (n = 144), or intravenous CS (n = 148). Twenty-five (6%) underwent colectomy at ≤1 year, 33 (9%) at ≤2 years, and 35 (13%) at ≤3 years. Further, 32/35 patients who had colectomy failed infliximab. An initial PUCAI ≥ 65 was highly associated with colectomy (P = 0.0001). A logistic regression model predicting colectomy using the PUCAI, hemoglobin, and erythrocyte sedimentation rate had a receiver operating characteristic area under the curve of 0.78 (95% confidence interval [0.73, 0.84]). Addition of a pretreatment rectal gene expression panel reflecting activation of the innate immune system and response to external stimuli and bacteria to the clinical model improved the receiver operating characteristic area under the curve to 0.87 (95% confidence interval [0.82, 0.91]). CONCLUSIONS A small group of children newly diagnosed with severe UC still require colectomy despite current therapies. Our gene signature observations suggest additional targets for management of those patients not responding to current medical therapies.

An immune gene expression signature distinguishes central nervous system metastases from primary tumours in non–small-cell lung cancer

BackgroundDissemination of non–small-cell lung cancer (NSCLC) in the central nervous system is a frequent and challenging clinical problem. Systemic or local therapies rarely prolong survival and have modest activity regarding local control. Alterations in gene expression in brain metastasis versus primary tumour may increase aggressiveness and impair therapeutic efforts. MethodsWe identified 25 patients with surgically removed NSCLC brain metastases in two different patient cohorts. For 13 of these patients, primary tumour samples were available. Gene expression analysis using the nCounter® PanCancer Immune Profiling gene expression panel (nanoString technologies Inc.) was performed in brain metastases and primary tumour samples. Identification of differentially expressed genes was conducted on normalized data using the nSolver analysis software. ResultsWe compared gene expression patterns in brain metastases with primary tumours. Brain metastasis samples displayed a distinct clustering pattern compared to primary tumour samples with a statistically significant downregulation of genes related to immune response and immune cell activation. Results from KEGG term analysis on differentially expressed genes revealed a concomitant enrichment of multiple KEGG terms associated with the immune system. We identified a 12-gene immune signature that clearly separated brain metastases from primary tumours. ConclusionsWe identified a unique gene downregulation pattern in brain metastases compared with primary tumours. This finding may explain the lower intracranial efficacy of systemic therapy, especially immunotherapy, in brain metastasis of patients with NSCLC.

Quantitative Transcriptional Biomarkers of Xenobiotic Receptor Activation in Rat Liver for the Early Assessment of Drug Safety Liabilities.

The robust transcriptional plasticity of liver mediated through xenobiotic receptors underlies its ability to respond rapidly and effectively to diverse chemical stressors. Thus, drug-induced gene expression changes in liver serve not only as biomarkers of liver injury, but also as mechanistic sentinels of adaptation in metabolism, detoxification, and tissue protection from chemicals. Modern RNA sequencing methods offer an unmatched opportunity to quantitatively monitor these processes in parallel and to contextualize the spectrum of dose-dependent stress, adaptation, protection, and injury responses induced in liver by drug treatments. Using this approach, we profiled the transcriptional changes in rat liver following daily oral administration of 120 different compounds, many of which are known to be associated with clinical risk for drug-induced liver injury by diverse mechanisms. Clustering, correlation, and linear modeling analyses were used to identify and optimize coexpressed gene signatures modulated by drug treatment. Here, we specifically focused on prioritizing 9 key signatures for their pragmatic utility for routine monitoring in initial rat tolerability studies just prior to entering drug development. These signatures are associated with 5 canonical xenobiotic nuclear receptors (AHR, CAR, PXR, PPARα, ER), 3 mediators of reactive metabolite-mediated stress responses (NRF2, NRF1, P53), and 1 liver response following activation of the innate immune response. Comparing paradigm chemical inducers of each receptor to the other compounds surveyed enabled us to identify sets of optimized gene expression panels and associated scoring algorithms proposed as quantitative mechanistic biomarkers with high sensitivity, specificity, and quantitative accuracy. These findings were further qualified using public datasets, Open TG-GATEs and DrugMatrix, and internal development compounds. With broader collaboration and additional qualification, the quantitative toxicogenomic framework described here could inform candidate selection prior to committing to drug development, as well as complement and provide a deeper understanding of the conventional toxicology study endpoints used later in drug development.

Association of immune microenvironment to response in treatment-naïve non-small cell lung cancer (NSCLC) samples with follow-up second-line immunotherapy data.

49 Background: Archival specimens collected months or years prior to starting immunotherapy are often used to identify patients for second line immune checkpoint inhibitor (ICI) treatment. PD-L1 expression and the immune microenvironment in these patients may have altered over time following multiple lines of failed standard of care (SOC) treatments. Methods: Formalin fixed paraffin embedded (FFPE) tumor samples, taken during resection performed as first line surgical treatment from a cohort of NSCLC patients (n = 18), were evaluated by Nanostring using the IO360 gene expression panel, and by immunohistochemistry (IHC) for CD3, CD8, PD-L1, CD68 and CD163. The resultant immune profiles were correlated with the clinical follow-up data for radiotherapy, SOC chemotherapy, and second line immunotherapy with the aim of understanding whether immune signatures predictive of response to ICI therapy may be identified in such samples. Results: Of the 18 cases, clinical follow-up data indicated objective response to ICI therapy for 4 patients, with the mean time from initial diagnosis to ICI treatment being 2.8 years (range: 0.4 to 8.5 years). Although pathologist PD-L1 IHC scores were not predictive of response, IHC image analysis data revealed significant increases in CD3 (2.3-fold) and CD8 (2.7-fold) T cell numbers in the responder population. In addition, although CD68+ macrophage frequencies did not differ significantly between responder and non-responder populations, reduced M2-like CD163+ macrophage/monocyte numbers were evident for responders. While the Tumor Inflammation Signature was not predictive of response, several gene expression signatures were significantly associated with response including increased abundance of CD8 T cells, cytotoxic cells, cytotoxicity, MHC class II antigen presentation and Melanoma-Associated Antigens (MAGE). Conclusions: Despite these patients having received various lines of radiotherapy and SOC chemotherapy prior to receiving immunotherapy, immune profiles associated with response to second line immunotherapy were detected in surgical first line resection samples.

Differential Diagnosis of Malignant Pleural Mesothelioma on Cytology: A Gene Expression Panel versus BRCA1-Associated Protein 1 and p16 Tests

Pleural effusions are among the first clinical manifestations of malignant pleural mesothelioma (MPM) and often constitute the only available material for diagnosis. Although an MPM diagnosis can be reliable on cytology, the reported sensitivity is low (30% to 75%). Particularly, it can be hard to discriminate epithelioid MPM, the most common histotype, from reactive mesothelial hyperplasia (MH). Currently, BRCA1-associated protein 1 (BAP1) and CDKN2A (p16), evaluated by immunohistochemistry and fluorescent in situ hybridization, respectively, are the most valuable markers to discriminate MPM and MH. Both markers have a high specificity, but their sensitivity is not always satisfying, even when used together. We have recently developed a 117-gene expression panel, based on Nanostring technology, able to differentiate epithelioid MPM from MH pleural tissues better than BAP1 and p16. Herein, we evaluated the efficacy of the same panel on an independent retrospective cohort of 23 MPM and 11 MH pleural effusions (cell blocks and smears). The overall sensitivity and specificity of the panel were equal to 0.9565 and 1, respectively. Moreover, the panel performance was compared with BAP1 and p16 on 25 cell blocks. Sensitivity levels of gene panel, BAP1 alone, p16 alone, and BAP1 plus p16 were 1, 0.5882, 0.4706, and 0.7647, respectively. Specificity was always 1. Although further validation is needed, this gene panel could really facilitate patients' management, allowing a definitive MPM diagnosis directly on pleural effusions.

Wnt Pathway Gene Expression Is Associated With Arterial Stiffness.

BackgroundAnimal and in vitro experiments implicate the Wnt pathway in cardiac development, fibrosis, vascular calcification, and atherosclerosis, but research in humans is lacking. We examined peripheral blood Wnt pathway gene expression and arterial stiffness in 369 healthy African ancestry men (mean age, 64 years).Methods and ResultsGene expression was assessed using a custom Nanostring nCounter gene expression panel (N=43 genes) and normalized to housekeeping genes and background signal. Arterial stiffness was assessed via brachial‐ankle pulse‐wave velocity. Fourteen Wnt genes showed detectable expression and were tested individually as predictors of pulse‐wave velocity using linear regression, adjusting for age, height, weight, blood pressure, medication use, resting heart rate, current smoking, alcohol intake, and sedentary lifestyle. Adenomatous polyposis coli regulator of Wnt signaling pathway (APC), glycogen synthase kinase 3β (GSK3B), and transcription factor 4 (TCF4) were significantly associated with arterial stiffness (P<0.05 for all). When entered into a single model, APC and TCF4 expression remained independently associated with arterial stiffness (P=0.04 and 0.003, respectively), and each explained ≈3% of the variance in pulse‐wave velocity.ConclusionsThe current study establishes a novel association between in vivo expression of the Wnt pathway genes, APC and TCF4, with arterial stiffness in African ancestry men, a population at high risk of hypertensive vascular disease.

The combination of WGS and RNA-Seq is superior to conventional diagnostic tests in multiple myeloma: Ready for prime time?

The diagnosis and risk stratification of multiple myeloma (MM) is based on clinical and cytogenetic tests. Magnetic CD138 enrichment followed by interphase FISH (fluorescence in situ hybridisation) is the gold standard to identify prognostic translocations and copy number alterations (CNA). Although clinical implications of gene expression profiling (GEP) or panel based sequencing results are evident, those tests have not yet reached routine clinical application. We set up a single workflow to analyse MM of 211 patients at first diagnosis by whole genome sequencing (WGS) and RNA-Seq and validate the results by FISH analysis. We observed a 96% concordance of FISH and WGS results when assessing translocations involving the IGH locus and an overall concordance of FISH and WGS of 92% when assessing CNA. WGS analysis resulted in the identification of 17 additional MYC-translocations that were missed by FISH analysis. RNA-Seq followed by supervised clustering grouped patients in their expected genetically defined subgroup and prompted the assessment of WGS data in cases that were not congruent with FISH. This allowed the identification of additional IGH-translocations and hyperdiploid cases. We show the reliability of WGS an RNA-Seq in a clinical setting, which is a prerequisite for a novel routine diagnostic test.

Minimal Residual Disease in Melanoma:molecular characterization of in transit cutaneous metastases and Circulating Melanoma Cells recognizes an expression panel potentially related to disease progression

Isolating circulating melanoma cells (CMCs) represents a powerful method to monitor minimal residual disease. We documented that MCAM/MUC18/CD146 expression is strongly associated with disease progression. ABCB5 is melanoma-stem antigen with self-renewal, proliferation, differentiation, tumorigenicity capabilities. These findings supported us to improve CMC detection, investigating MCAM/MUC18/CD146 and ABCB5 as enrichment targets in MM progression. Moreover, we decided to compare possible molecular diversity of these CMC fractions with metastatic tissue expression, collecting concomitantly cutaneous in transit metastases (CTM). We enriched CMCs from eight melanoma patients staged ≥pT1b AJCC, who developed CTMs at baseline or during follow up. We assessed a gene expression panel comprising ABCB5, the differentiation markers (Tyrosinase, MART1), angiogenic factors (VEGF, bFGF), the cell-cell adhesion molecules (MCAM/MUC18/CD146 5′-portion, Long, and Short isoforms, E-Cadherin, N-Cadherin, VE–Cadherin) and matrix-metallo-proteinases (MMP2 and MMP9) via high-sensitive RT-PCR. Preliminary findings defined three distinct sub-populations: “endothelial” CD45-CD146+CMCs, “stem” CD45-ABCB5+CMCs and a “hybrid- stem-endothelial”- CD45-MCAM+ABCB5+CMCs. The expression panel documented that – almost high expression found in CTMs – like in 73.5% of CMCs resulted positive for at least one transcript at baseline, showing gene-expression variability. Longitudinal monitoring documented shut-down of all gene-expressions in “endothelial”- and “hybrid stem-endothelial”-subsets, whilst persistency or acquisition of MCAM/MUC18/CD146, VE-CADH and MMPs was documented in disease-progression status.Conversely, a drastic expression shut-down was documented when patients achieved clinical remission. The “stem”- CMCs fraction” showed quite lower gene expression frequencies. MCAM/MUC18/CD146 and ABCB5 as melanoma-specific-targets are effective in the selection of highly primitive CMCs and highlights those putative genes associated with disease spreading progression.

Immunological signature meta-analysis across lung cancer cohorts within the NanoString Clinical Transcriptional Atlas Group (CTAG) associated with patient outcome and history

Abstract Background With the approval of anti-PD1 blocking antibodies in a growing number of indications, understanding the mechanisms responsible for potentiating response to these agents has been a critical avenue of research. However, most studies rely on the use of single cohorts and are relatively limited in their scope, thus limited their utility. Methods We created a consortium of investigators who share clinical and accompanying transcriptional data collected on the NanoString® nCounter® platform from patients treated with single agent immunotherapy to facilitate biomarker research. In this study, we apply a meta-analysis to a combined cohort of 4 lung cancer studies (n = 150) to identify transcriptional correlates of response to identify patients who are likely to experience clinical benefit. Results RNA was profiled with the NanoString IO360 and IO360 beta gene expression panels, and 43 signatures that describe facets of the immune response, tumor biology, and the tumor microenvironment were calculated. Included in these signatures is the Tumor Inflammation Signature (TIS), an investigational 18 gene signature of a suppressed adaptive immune response which enriches for clinical response to pembrolizumab. These signatures, as well as genes of interest identified in a given cohort, were compared to objective response, overall survival, or progression free survival across the cohorts. We confirm previously reported association of TIS with patient outcome and identify immune cell subsets that are also associated with response. In addition, we examine differences between patients with distinct mutational backgrounds, smoking histories, or histological classifications, as well as differences attributable to biopsy location. Conclusion This multi-cohort study allows for the development of multi-variate predictors of response to anti-PD1 monotherapy using a single research assay to transcriptionally profile the tumors. We can generate robust predictions from real-world cohorts, which may lead to the development of improved diagnostic assays to guide treatment decisions. Legal entity responsible for the study NanoString Technologies. Funding NanoString Technologies. Disclosure N. Radosevic-Robin: Research grant / Funding (institution): NanoString Technologies. J. Reeves: Full / Part-time employment: NanoString Technologies. K. Leroy: Research grant / Funding (self): NanoString Technologies. M. Duruisseaux: Research grant / Funding (self): NanoString Technologies. P. Morel: Full / Part-time employment: NanoString Technologies. M. Bhagat: Research grant / Funding (institution): NanoString Technologies. F. Penault-Llorca: Advisory / Consultancy, Research grant / Funding (institution): NanoString Technologies. D. Damotte: Research grant / Funding (institution): NanoString Technologies. F. Goldwasser: Research grant / Funding (institution): NanoString Technologies. A. Brindel: Research grant / Funding (institution): NanoString Technologies. M. Cumberbatch: Research grant / Funding (institution): NanoString Technologies. S. Ong: Full / Part-time employment: NanoString Technologies. J. Lopez: Research grant / Funding (institution): NanoString Technologies. S. Warren: Full / Part-time employment: NanoString Technologies.

Analysis of the immune microenvironment in pre-treatment non-small cell lung cancer (NSCLC) patients with follow-up response data to second-line immunotherapy

Abstract Background Patients selected based on PD-L1 expression for second-line immune checkpoint inhibitor (ICI) treatment are often identified using archival specimens collected months or years prior to starting immunotherapy. Such patients may have received and failed multiple lines of standard of care (SOC) treatments with subsequent potential impact on PD-L1 expression and immune microenvironment. Methods We have analysed formalin fixed paraffin embedded (FFPE) tissues in a cohort of NSCLC patients (n = 16) taken during resection performed as first-line surgical treatment for which radiotherapy, SOC chemotherapy, and second-line immunotherapy clinical follow-up data are available. The immune microenvironment was evaluated by immunohistochemistry (IHC) plus digital image analysis for CD3, CD8, PD-L1, CD68 and CD163, and by Nanostring using the IO360TM gene expression panel, with the aim of exploring whether immune signatures predictive of response to ICI therapy may be identified in such samples. Results Clinical follow-up data indicated objective response to ICI therapy for 4/16 patients (n = 2 Nivolumab, n = 1 Pembrolizumab, n = 1 Durvalumab) with time from first diagnosis to receiving second-line ICI treatment ranging from 5 to 102 (mean 33.6) months. During this time these patients received various lines of radiotherapy and SOC chemotherapy prior to receiving immunotherapy. While the Tumor Inflammation Signature was not predictive of response, gene expression analysis did identify several signatures associated significantly with response, including increased abundance of CD8 T cells, cytotoxic cells, cytotoxicity, MHC class II antigen presentation and Melanoma-Associated Antigens (MAGE). These data were supported by a significant elevation of CD8 T cells by IHC in the responder versus non-responder populations, from a mean of 329 to 961 CD8+ T cells/mm2. Conclusion Taken together these data demonstrate that despite various lines of previous radiotherapy and chemotherapy spanning several years, immune profiles associated with response to second-line immunotherapy can be detected in surgical first-line resection samples. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure M. Bhagat: Shareholder / Stockholder / Stock options, Officer / Board of Directors: TriStar Technolgoy Group. All other authors have declared no conflicts of interest.

126P - Predictors of response to checkpoint inhibitors in naïve and ipilimumab-refractory melanoma

BackgroundResponse to checkpoint inhibitors (CI) is governed by the tumor immune environment and understanding this immune contexture can predict response. Therapeutic intervention can change this environment even in the absence of clinical response. Patients failing initial immunotherapy may respond to a second line of CI; however, these cohorts show lower overall response rates (ORR). This study identifies transcriptional signatures associated with response to first- and second-line CI monotherapy in melanoma. MethodsCI-naïve or ipilimumab-refractory patients were treated with ipilimumab, nivolumab or pembrolizumab at the Instituto Nazionale Tumori and clinical response was evaluated by irRECIST 1.1 criteria. Pretreatment tumor biopsies (n=82) from metastatic lesions were collected and RNA was profiled with the NanoString® IO360 gene expression panel. ResultsCompared to CI-naïve cohorts, ipilimumab-refractory cohorts had reduced ORR to nivolumab (naïve: 35%, n=6; refractory: 20%, n=10) or pembrolizumab (naive: 67%, n=6; refractory: 20%, n=10) with multiple genes differentially expressed between groups. The Tumor Inflammation Signature, an investigational 18 gene signature of suppressed adaptive immune response enriching for pembrolizumab response, was higher in responders versus non-responders in first-line (log2 fold change: 1.56, p=0.21), but not second-line pembrolizumab (log2 fold change: 0.41, p=0.60). First-line pembrolizumab responders had elevated MHC2 (log2 fold change: 1.35, p=0.02) and B cell (log2 fold change: 2.14, p=0.02) signatures. Upon stratifying the CI-naïve cohort between no prior treatment versus prior targeted/chemotherapy, the latter had increased immune expression suggesting these therapies prime the tumor immune environment. ConclusionsCorrelating patterns of tumor gene expression with clinical response can lead to the development of biomarkers enriching for CI response in both first-line and CI-refractory patients. Utilization of a clinical grade platform such as the NanoString nCounter® may speed the development of diagnostic assays used to predict and monitor patient response to immunotherapy. Legal entity responsible for the studyThe authors. FundingNanoString Technologies. DisclosureS. Ong: Full / Part-time employment: NanoString Technologies. S. Warren: Shareholder / Stockholder / Stock options, Full / Part-time employment: NanoString Technologies. A. Cesano: Shareholder / Stockholder / Stock options, Full / Part-time employment: NanoString Technologies. J.M. Beechem: Shareholder / Stockholder / Stock options, Full / Part-time employment: NanoString Technologies. P.A. Ascierto: Advisory / Consultancy: Amgen; Advisory / Consultancy: Array; Advisory / Consultancy: BMS; Advisory / Consultancy: Incyte; Advisory / Consultancy: Immunocore; Advisory / Consultancy: MedImmune; Advisory / Consultancy: IDERA; Advisory / Consultancy: Genmab; Advisory / Consultancy: Merck; Advisory / Consultancy: Roche; Advisory / Consultancy: Genentech; Advisory / Consultancy: Sandoz; Advisory / Consultancy: Syndax; Advisory / Consultancy: Sun Pharma; Advisory / Consultancy: Ultimovacs; Advisory / Consultancy: Pierre Fabre; Advisory / Consultancy: Novartis. All other authors have declared no conflicts of interest.