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Table 1 Approaches for measuring peripheral biomarkers

From: Peripheral immune-based biomarkers in cancer immunotherapy: can we realize their predictive potential?

ApproachSampleStrengthsManufacturers and/or examples of technologies
Whole transcriptome profiling, RNA- seq, single-cell RNA-seqRNA from PBMCsRNA-seq
• Fast and high efficiency
• Broad, dynamic range
• Detects differentially expressed genes
• Measures average expression level
• Uses millions of short reads (sequence strings), so all RNA in a sample can be investigated
• Illumina
  Single-cell (scRNA-seq)
• Measures the distribution of expression levels for each gene
• Expression patterns of individual cells can be defined in complex tissues
• High resolution of cell-to-cell variation
• Bio-Rad® single-cell RNA-sequencing solution
• 10X Genomics
Epigenetic differentiation-based immune-cell quantificationGenomic DNA from fresh or frozen whole blood, PBMCs• Broad range of acceptable sample conditions (e.g. samples can be frozen and shipped without other steps)
• Standardized measurements and circulating and tissue-infiltrating immune cells can be compared as an alternative to flow cytometry for peripheral blood samples and IHC for solid tissues
• Quantitative real-time PCR-assisted cell counting
Chromosomal confirmation signaturesBlood• CCSs can provide a stable framework from which changes in the regulation of a genome can be analyzed• EpiSwitch™
Protein microarrayFresh or frozen serum and plasma• Versatile and robust platform
• Miniaturized features, high throughput, and sensitive detections
• Reduction in sample volume used
• Variety of biological samples can be analyzed
• ProtoArray® (Life Technologies) can analyze serologic response of 9000 proteins simultaneously
• SOMAscan® Assay
• Olink Proteomics
Mass spectrometryBlood• Mass spectrometry-based protein measurements in blood• Biodesix
Flow cytometryBlood, fresh or frozen PBMCs, circulating tumor cells• Multiparameter measurements at single-cell level
• Rapid, high throughput manner
• Cytometers available at reasonable cost
• Recent advances in lasers/fluorochrome technology allows multiparameter analysis of rare cells (e.g. tumor antigen-specific T lymphocytes)
• BD LSRFortessa™ X-20
• BD FACSymphony™
Mass cytometryBlood, fresh or frozen PBMCs• Multiparameter single-cell analysis
• Heavy metal ions as antibody labels overcome limitations of fluorescence-based flow cytometry
• Little overlap between channels and no background (up to 40 labels per sample)
• Increased number of phenotypic and functional markers can be probed
• Comprehensive analysis of profile and function of immune populations (e.g. time-of-flight cytometry by Helios™)
T- and B-cell receptor deep sequencingPBMCs formalin-fixed paraffin-embedded• Identify changes in T- & B-cell populations, both in circulation and within tumors
• Millions of T- & B-cell receptor sequences can be read from a single sample
• Identify clonal expansion (measure of adaptive immune response)
• Has been used to show clinical response to cancer immunotherapy
• ImmunoSEQ® immune profiling system at the deep level (Adaptive Biotechnologies)
• Illumina HiSeq system
ELISA and multiplex assaysBlood• Widely used
• Multiple biomarkers measured at once
• Small sample volume
• Measures soluble mediators (e.g. cytokines, chemokines, autoantibodies)
• Multianalyte immunoassays: Simple Plex™ MesoScaleDiscovery Luminex, Quanterix™
  1. CCS Chromosomal confirmation signature, ELISA Enzyme-linked immunosorbent assay, IHC Immunohistochemistry, PBMC Peripheral blood mononuclear cell, PCR Polymerase chain reaction