RNA Sequencing (RNA-seq)
RNA-seq takes advantage of next-generation sequencing to study transcriptome profiling, including quantity, quality of RNA sequences, alternatively spliced transcripts, gene fusions, or mutations. As such, it allows for monitoring of gene expression over time, or differences in gene expression in different treatment groups. The increasing use of RNA-seq in clinical and basic science settings provides a powerful tool to assess novel biomarkers in the tumor microenvironment (TME). Advanced computational methods are making it possible to resolve the composition of the tumor immune infiltrate, infer the immunological phenotypes of cells, and assess the immune receptor repertoire in RNA-seq data. These immunological characterizations have increasingly important implications for guiding immunotherapy use.
Gene expression signatures yield a large amount of information that can be used for diagnosis, prognosis, or prediction of the therapeutic response. These powerful assays are harnessing the diversity and adaptability of the immune system and hold significant promise for the personalized treatment of cancer. The PanCancer IO 360 is a research panel for gene expression measurement designed to evaluate variables relevant to mechanisms of immune response/evasion that can potentially be modulated through therapeutic intervention. The IO360 panel supports the development of signatures to potentially predict a patient response to a variety of immunotherapeutic interventions.
Whole Exome Sequencing (WES)
WES allows identification of the genetic variants in all exons of an individual genome. WES is an efficient method for unbiased detection of common or rare somatic mutations and copy number variants associated with cancer. WES allows comprehensive measurement of tumor mutational burden (TMB) and is a measure of the total number of somatic coding mutations in a tumor. Accumulating evidence suggests potential usefulness of TMB and tumor neo-antigen loads based on WES that are currently being investigated as potential biomarkers of response to cancer immunotherapy.
TCR-Seq assays can be used to characterize T-cell receptor (TCR) repertoire before and after therapy to study how TCR diversity is associated with clinical response. CIMACs use Adaptive Biotechnologies’ immunoSEQ Technology to identify and enumerate the repertoire of T cells, as well as B cells, within a sample. ImmunoSEQ Assays utilize a multiplex PCR-bias controlled assay that enables sequencing directly from genomic DNA or cDNA from various sample types, including FFPE and blood. Researchers can track specific T- or B-cell clones across sample types longitudinally and monitor immune repertoire dynamics, to study therapeutic mechanism of action and identify potential biomarkers of response to therapy. Note: immunoSEQ is For Research Use Only. Not for use in diagnostic procedures.
In addition to the immunoSEQ assay, CIMACs also use RNA-based TCRseq for bulk RNA and single cell TCR sequencing in selected trials. This assay can be used to detect paired alpha/beta TCR clonotypes in a single cell or to perform alpha and beta analysis in bulk RNA samples for repertoire analysis. It can be performed with a limited amount of input material.
Single cell TCR-sequencing (scTCR-seq), including single-cell-targeted RNA-seq, combines information about T cell phenotype and T cell clonality. It is applicable to tumor-infiltrating T cells, or to sorted populations of antigen-specific T cells. This assay is used to explore whether dominant clones have a similar or heterogeneous phenotype and whether phenotype segregates with TCR clonality.
HLA-Seq: Epitope Prediction
Circulating Tumor DNA (ctDNA)
ctDNA may reflect the entire tumor genome diversity; it has gained traction for its potential clinical utility as “liquid biopsies” measured in plasma or serum to monitor tumor progression, predict treatment response or relapse, or determine target driver mutations. Both WES and targeted approaches using histology-specific gene panels are standardized and are available at CIMACs for ctDNA detection.
Assay for Transposase-Accessible Chromatin (ATAC-seq)
The ATAC-seq assay with high throughput sequencing facilitates “regulome” analysis. The readout is a comprehensive map of chromatin accessibility and gene regions that are open for transcription in the cell type analyzed. This assay can be used to evaluate the epigenomic determinants of responsiveness to treatment.
HTG-EdgeSeq (gene expression)
HTG EdgeSeq™ panels provide comprehensive tumor profiling of immune response and systems biology. The HTG EdgeSeq Precision Immuno-Oncology Panel characterizes the immune response in tumor microenvironments, while the HTG EdgeSeq Oncology Biomarker Panel allows for a thorough interrogation of the tumor.
Twenty-three immunophenotyping signatures, including tumor inflammation and stromal response to a tumor, are part of HTG’s EdgeSeq Reveal software. These signatures can be applied within the software for rapid gene expression visualization. The HTG EdgeSeq Reveal Oncology Signatures are built upon the HTG EdgeSeq Precision Immuno-Oncology Panel and developed to enable researchers to understand the tumor microenvironment better.
Microbiome (16S Deep Sequencing)
16S rRNA amplicon sequencing provides the relative or absolute abundance of microbes in a biological sample. Microbial DNA and the V4 hypervariable region of the 16S rRNA gene is amplified by PCR, sequencing is run, and computational analysis is performed to determine relative abundances of taxa, alpha-, and beta diversity, and association taxa with phenotypes. Characterization of the microbiome may help to stratify immunotherapy responders from non-responders, contribute to the development of microbiome-based combination therapy to improve response rate, and/or reduce adverse events, such as colitis.
Single-cell RNA-sequencing (scRNA-seq)
scRNA-seq is an RNA-seq performed on isolated cells instead of a bulk of thousands to millions of cells. This assay is used to elucidate complex phenotypic and functional characteristics of heterogeneous immune populations. It helps to provide valuable insights into disease mechanisms of tumor progression and therapeutic responses.
Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq)
CITE-Seq allows the combination of single-cell-level RNA sequencing with quantitative and qualitative analysis of surface proteins using antigen-specific monoclonal antibodies.