Each Cancer Immune Monitoring and Analysis Center (CIMAC) provides a wide range of state-of-the-art analyses for genomic, phenotypic, and functional assays using analytically validated platforms and, for core assays, harmonized protocols to allow comparison of datasets across CIMAC sites and studies. More than one CIMAC laboratory may collaborate on a single trial to provide multiple specialized assays.

Click the links below to view the available SOPs for each assay listed.

New assays continue to be added by the network. This page will be updated with additional information as it becomes available.

Multiplex Immunohistochemistry/Immunofluorescence (mIHC/IF)

mIHC and IF allow spatial mapping and quantification of target molecules simultaneously by using highly sensitive and specific antibodies on a single tissue section. The main advantages of this method over singleplex IHC are tissue preservation for patients with limited sample availability, being able to demonstrate co-expression of multiple markers, and spatiotemporally mapping all targets showing the cross talk between different cell types and proteins on the same tissue section. Because mIHC/IF allows for comprehensive phenotypic characterization of tumor microenvironment (TME) and functionality assessment, quantity and/or spatial distribution of individual or co-expressed markers can be found as predictive and/or prognostic biomarkers at baseline and for monitoring response to immunotherapy.

Singleplex Chromogenic IHC (sIHC)

sIHC is the gold standard of immunohistochemistry assays; it detects one antigen target at a time on a single tissue section. Besides its use as clinical and companion diagnostics, it is also a reliable control assay for different antibodies used with mIHC/IF. Given that mIHC/IF, including different panels of antibodies, is available at CIMACs as a Tier 1 assay, singleplex IHC assays will primarily focus on measurements of PD-L1 expression as a biomarker of one of the most important checkpoint molecules of the tumor that predicts immunotherapy response. FDA-approved companion or complementary PD-L1 assay kits matching different checkpoint therapeutic agents will be used.

Multiplexed Ion Beam Imaging (MIBI)

MIBI uses secondary ion mass spectrometry to image targets using antibodies tagged with heavy metal reporters and yields a mass spectrometry readout. It can analyze up to 100 targets simultaneously, can provide new insights by integrating tissue microarchitecture with highly multiplexed protein expression patterns, and can offer valuable information for basic research, as well as clinical diagnostics.

Mass Cytometry (CyTOF)

CyTOF is a high-dimensional version of flow cytometry that uses heavy-metal ion tags detected by mass spectrometry in place of fluorescent labels. The readout of this technology results in signals for each individual cell that correlate with the expression of the target proteins, function, and phenotypes across a detailed spectrum of cell types (e.g., CD8, CD4 T cells, regulatory T cells, B cells, NK cells, myeloid cell subsets, etc.). CyTOF allows for the quantifying of the frequencies of immune subsets and markers of immune activation or suppression over time of disease progression or response to therapy.

In addition to allowing detailed characterization of cell populations on the basis of surface receptor expression patterns, CyTOF proteomics can also be used to evaluate signaling pathways using antibodies that target phosphorylated protein epitopes. This approach, referred to as phospho-CyTOF, can offer a detailed dynamic characterization of the nature of immune activation to comprehensively evaluate single-cell signatures of activation across all major circulating immune subsets.

High-Dimensional Flow Cytometry


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/BCR Clonality

TCR-Seq assays can be used to characterize clonality of the T-cell receptor (TCR) repertoire before and after therapy to study how TCR diversity is associated with clinical response. 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


Neoantigen 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)

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.

O-link is a technique for detection and quantification of soluble proteins. It can be used for the profiling of cytokines, chemokines, and growth factors, as well as the detection of circulating immune co-stimulatory and inhibitory molecules. Cytokines are key factors regulating immune response and intercellular communication in the tumor microenvironment. As such, cytokines possess diagnostic and prognostic potential, and cytokine production may reflect effects of immunotherapies. Cytokines may also be used in cancer therapy and to modulate immune response.


ELISA/Grand Serology

Grand Serology is an ELISA-based assay using arrays that consist of a very large number of targets that can be simultaneously measured with high throughput to characterize the repertoire of serological responses in the serum of cancer patients. The profiles can have diagnostic significance and measure changes in antibody responses to treatments, including immunotherapies.

MesoScale Discovery

Longitudinal reference materials

Reference materials for longitudinal monitoring of core assay performance across the CIMAC-CIDC Network have been obtained from commercial and NCI-supported sources and include the following:

  • Custom-activated peripheral blood mononuclear cells to serve as reference material for CyTOF
  • HapMap cell line samples to serve as reference materials for genomic studies
  • Tissue microarrays (TMAs) from the NCI-supported Cooperative Human Tissue Network to serve as a master reference for all tissue imaging (e.g., IHC/IF) studies