Abstract 4560: A cost-effective and rapid assay for profiling of immunobiology and the development of predictive signatures for response to immunotherapies
Authors: Peilin Chen, Sandy Spurgeon, Jing Wang, Michael Gonzales, Lianne McLean, Tom Goralski
Journal: Cancer Research (2018)

Abstract Introduction: Gene expression profiling is an important tool to monitor the human immune response to different antitumor agents for drug development and to identify predictive signatures for response to immunotherapies. We have developed and analytically validated a gene expression assay panel, namely the AdvantaTM Immuno-Oncology Gene Expression Assay (AdvantaTM I/O GE Assay), on the Fluidigm BiomarkTM HD system, a microfluidic real-time PCR system, for investigation of antitumor agent effects on immune inhibitory pathways. The assay panel consists of 170 unique gene targets in categories such as T cell subset markers (e.g., CD4), cytokines and chemokines (e.g., IFNg), immune regulation (e.g., PD-L1, PD-L2) and immune cell-fate markers (e.g., NK) associated with immune-checkpoint inhibitory pathways for cancer treatments. Method: The Assay consists of Fluidigm Dynamic Array™ IFCs, an assay panel and RT-PCR reagents. The assay panel is composed of TaqMan™ gene expression assays provided in two 96-well plates: 96 assays on one and 79 assays on another with 17 unused assay wells available for customization. Each plate contains five housekeeping genes for assay normalization. This assay configuration allows customers to test 96 samples on two Fluidigm 96.96 Dynamic Array™ IFCs. Two primer pools for preamplification, and a positive control have been developed along with a 24.192 Dynamic Array™ IFC which enables 24 samples and the complete panel, 170 genes to be run on a single IFC. The assay has been analytically validated by a third party (Q2/EA) on the 96.96 IFC. This study is to verify the assay and kit reagents on the 24.192 IFC. A universal RNA was reversely transcribed into cDNA and pre-amplified with the preamp primer pools for all 170 genes. Three tissue cDNAs (lymph node, brain and Jurkat cells) and the positive control, which contains all 170 targets, were used to evaluate amplification efficiency, linearity and reproducibility. Each sample was tested in three replicates on 24.192 IFCs in six independent runs. Results: The results show an average amplification efficiency across all assays of 98.2% and linearity (RSQ) 0.999 for universal RNA. The efficiency and linearity for the positive control are 0.999 and 0.998, respectively. The within-run replicate correlation (RSQ) is 0.997 for the universal RNA, 0.992 for Jurkat cDNA, 0.986 for brain cDNA, and 0.990 for lymph node cDNA. The between-IFC replicate correlation (RSQ) is 0.981 for universal RNA, 0.982 for Jurkat cDNA, 0.972 for brain cDNA and 0.977 for lymph node cDNA. Gene expression signatures for individual tissues were observed. Conclusions: The AdvantaTM I/O GE Assay on 24.192 IFC and the Fluidigm BiomarkTM HD system provides high amplification efficiency, excellent linearity and reproducibility and reduces the cost of routine laboratory testing for both immune checkpoint research and drug development. Citation Format: Peilin Chen, Sandy Spurgeon, Jing Wang, Michael Gonzales, Lianne McLean, Tom Goralski. A cost-effective and rapid assay for profiling of immunobiology and the development of predictive signatures for response to immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4560.