Applied Biosystems Genetic Analysis Virtual Conference

Cancer immunotherapy research is rapidly expanding our knowledge of tumor pathologies. In this talk, we will describe how Applied Biosystems™ TaqMan® gene expression panels, which query sets of genes important in tumor immuno-response, can be used to investigate colon tumor biology. We also analyzed microsatellite instability (MSI) using tumor and normal adjacent samples with the Applied Biosystems™ TrueMark™ MSI Assay. We analyzed the same samples with a TaqMan panel for T cell-inflamed gene expression and an anti-CTLA-4 resistance associated MAGE-A (CRMA) panel. To facilitate these analyses, we used Applied Biosystems™ TaqMan® Fast Advanced Master Mix and RFID-tagged pre-spotted plates on the RFID-enabled Applied Biosystems™ QuantStudio™ 6 Pro Real-Time PCR System. We correlated these expression patterns with MSI status and with PD-L1 antigen expression. Finally, we demonstrate how using RFID-tagged pre-spotted TaqMan Array plates that are run on QuantStudio 6 and 7 Pro Systems, followed by data analysis on Thermo Fisher Connect RQ gene expression analysis app, can streamline the workflow and reduce errors.

For Research Use Only. Not for use in diagnostic procedures.

Immuno-oncology (I-O) is the study of the ways in which body’s own immune system can be used to fight cancer. I-O research aims to develop cancer immunotherapies by enabling the adaptive immune system to recognize and specifically attack cancer cells while leaving healthy ones undamaged. I-O research can potentially uncover ways to enable immunogenicity of all types of cancer and facilitate long lasting, protective immunity against future recurrence. Immunotherapy is considered revolutionary in treating cancers and has become a standard of care along with chemotherapy, radiation, and surgery. Although immunotherapies have shown tremendous success in treating cancers, response rate to these agents is low and often associated with side effects. It is therefore important to find biomarkers which would predict response to immunotherapy with minimal side effects. Biomarker discovery in the immunotherapy space is complicated by the complex interactions between tumors and the immune system and by host and tumor variability. Many bioinformatics tools and new genomic and proteomic technologies are required to predict specific tumor signatures.

In this presentation we discuss the genetic analysis research tools available from Applied Biosystems and Ion Torrent to study biomarkers associated with immune check point inhibitors, adoptive T-cell transfer, and therapeutic cancer vaccines.

For Research Use Only. Not for use in diagnostic procedures.

The study of human development and diseases relies on analysis of samples obtained and manipulated ex-vivo. It is critical in such studies to know the provenance and confirm that the identity of these materials is correct. Furthermore, it is critical to confirm that once manipulated, these cells have the desired differentiated characteristics and have maintained genome integrity. In this study, we describe a complete workflow for quality control of ex-vivo manipulated human cells, including cell lines, induced pluripotent stem cells (iPSC) and chimeric receptor T-cells (CAR-T). Analysis of short tandem repeats (STRs) was used as a molecular “fingerprint” to track and confirm the identity of the manipulated cells. Pluripotency of iPCS was determined by analyzing the expression of a panel of differentiation-state specific genes. Genomic integrity was analyzed by molecular karyotyping using high-resolution microarrays. The presence or absence of pathogenic oncogenes in the manipulated cells was determined by sequencing a panel of oncogenic hotspot loci. Together, these results and robust workflows provides a comprehensive quality control of human cells, critical for the establishing clinical research-compliant ex-vivo manipulation methods.

The Clariom GO Screen Assay is a transcriptome profiling solution that provides a high-throughput, low-cost method to directly measure the expression of ~20,000 genes. With relevant probes derived from the Gene Ontology (GO) Consortium and content developed in collaboration with pharmaceutical researchers, this assay helps you quickly assess changes in key genes and pathways. Whether you want to accelerate the development of new drug compounds, reduce reliance on animal testing for toxicological research, examine cellular response to cosmetic or skincare formulations, or rapidly identify immune-response pathways involved in novel infectious diseases such as SARS-CoV-2, Clariom GO Screen Assay is a reliable, low-cost platform that speeds your time from experiment to analysis.

The TAILORx clinical trial showed that up to 70% of patients with breast cancer with estrogen receptor (ER+) do not benefit from chemotherapy. However, these patients continue to develop recurrences when treated with endocrine therapy alone. The addition of CDK4/5 inhibitors to endocrine therapy results in significant (8-12 months) delay in progression of disease. There is critical need to understand the mechanisms of resistance and development of novel targets/therapies for prevention of endocrine resistance and recurrences of ER+ breast cancer. We have recently demonstrated that Epithelial Splicing Regulatory Protein 1 (ESRP1), an epithelial cell-specific RNA binding protein and splicing factor, is associated with poor prognosis and endocrine resistance in human ER+ breast tumors (Gökmen-Polar, EMBO Reports 2019). We documented a novel role for ESRP1 in altering metabolic pathways by altering both gene expression and alternative splicing events. ESRP1 silencing further impacted cell/tumor growth and metabolism significantly. CDK4/CDK6 inhibitor effectively decreased the expression of proliferation pathway significantly, but did not block the metabolic alterations. Here we seek to investigate associated molecular mechanisms by performing RNA Binding Protein (RBP) immunoprecipitation followed by Clariom D transcriptomics microarray (RIP-Chip) to identify mRNA binding partners of ESRP1. RIP-Chip analysis identified PHGDH, a key metabolic enzyme, as a binding partner of ESRP1 in ER+ breast cancer. Binding was further validated using RNA electrophoretic shift assay (REMSA) against 5’ UTR probes and RIP-RT-qPCR. Further functional characterization in vitro showed that ESRP1 posttranscriptional regulates PHGDH by affecting its mRNA stability. Seahorse metabolic analyses of the PHGDH knockdown cells significantly reduced oxygen consumption rate (P<0.04) mimicking the effects of ESRP1 knockdown. In conclusion, this study documents a non-mutational pathway in mRNA and protein down regulation and altered cellular metabolism. We anticipate that the insights garnered from these studies will enable a more precise molecular understanding of metabolic pathways of endocrine resistance and facilitate discovery of new therapeutic targets for treatment of endocrine resistance.