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Table 3 Currently available translational biomarker assays for immune checkpoint inhibitors

From: Current status and perspectives in translational biomarker research for PD-1/PD-L1 immune checkpoint blockade therapy

Biospecimen Method Tissue/cell types Pros Cons References and recommended reading
FFPE IHC Tumor cells or tumor infiltrating immune cells Direct detection; accurately pinpoint cancer cells; highly sensitive; simplicity; low cost Requirement of trained pathologists; inconsistency for criteria used to score tumors such as PD-L1-positive or negative Herbst R (2014) [145]; Loughlin PA (2007) [164]
Multicolor IHC Tumor cells or tumor infiltrating immune cells Broad dynamic range; capability for multiplexing using different fluorescence channels; >10 protein targets are identified in the same sample; amenability for co-localization studies Absence of rigorous quantitative tests; limitation in some biomarker-driven clinical trials; user must select combinations of dyes Carvajal-Hausdorf DE (2014) [165]
T cell receptor deep sequencing TILs T cell count information;
T cell clonality in tumor
Heterogeneous expression of TIL Robbins HS (2013) [100]
Whole exome sequencing (WES) Tumor cells Characterization of tumor mutation load including nucleotide substitutions; structural rearrangements and copy number alterations; identification of the neoantigens and neoepitopes; affordable cost Require high-performance deep sequencing, computational bioinformatics support;
The pipelines are still at early developmental phase
Snyder A (2014) [104]; Rizvi NA (2015) [105];
Bouffet E (2016) [107];
Chen K-H (2016) [166]
Hugo W (2016) [106]
Blood ELISPOT assays (IFNγ and granzyme B) T cells in PBMCs Detection of tumor antigen-specific CD4+ and CD8+ T cell response with good assay sensitivity;
Relatively well validated assay
A poor correlation with clinically relevant immune responses Shafer-Weaver K (2006) [167]; Janetzki S (2008) [95] Malyguine A (2012) [94]; Janetzki S (2015) [93];
Flow cytometry (tetramer, polyfunctional analysis) T cells in PBMCs Assessment of tumor antigen-specific CD4+ and CD8+ T cells response; measure multiple functions; detection of neoantigen-specific CD8 + PD-1+ T cells; minimally invasive Merely in lab research, not as routine clinical monitoring yet Yuan J (2008) [84]; Attic S (2011) [85]; McNeil LK (2013) [86]; Barrera L (2015) [168]; Gros A (2016) [118]
Flow cytometry phenotype staining Whole blood immune phenotype Analyses of the frequency and proliferation of different subsets of immune cells; routine operation Dedicated resource and staff to perform the analyses Streitz M (2013) [169]; van Dongen JJ (2012) [170]
RNA-Seq (NGS) T cells in PBMCs Identification of genetic variants; a broader dynamic range; detection of more differentially expressed genes; fast and high efficiency More expensive than microarray; more complex for analysis; bulk signature, not single cell signals; need more validation Zhao S (2010) [171]
qPCR assay T cells in PBMCs High specificity; able to detect the reactivity of low-frequency T cells in the peripheral blood of metastatic cancer patients Bulk signature, not single cell signals; need more validation Kammula US (2008) [172]
Flow cytometry CTCs Qualitative analysis at the single cell level in a relatively short period of time; decrease the amount of blood needed; provide valuable information regarding the frequency, phenotype and/or the functionality of T cells Expensive; need more validation Zaritskaya L (2010) [83]
Cell sieve microfiltration assay and QUASR technique CTCs PD-L1 levels from CTCs or CAMLs serves as a surrogate for PD-L1 expression in tumor; as a marker for immunotherapy response Limited in lab research; need more validation Steven HL (2015) [173]; Adams DL (2014) [174]
  1. Abbreviations: FFPE formalin-fixed paraffin-embedded, PD-L1 program death-1, TIL tumor-infiltrating lymphocytes, IHC immunohistochemistry, CAMLs cancer-associated macrophage-like cells, ELISPOT enzyme-linked immunospot assay, CTL cytotoxic T-lymphocytes, CTCs circulating tumor cells, PBMC peripheral blood mononuclear cell, WES whole exome sequencing, NGS next-generation sequencing