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Last Updated: 08/27/2020

Clinical Pharmacodynamic Biomarker Assays

Pharmacodynamic (PD) Biomarker Assays developed by the DCTD Clinical Pharmacodynamic Program (CPP) are intended to investigate drug and downstream drug-induced effects. Validated biomarker assays are available to the scientific community through either the National Clinical Laboratory Network (NCLN) or CPP’s on-site training and critical reagent support.

NCLN-Supported Validated Biomarker Assays: Overview

The National Clinical Laboratory Network (NCLN) offers validated, harmonized SOPs while implementing uniform assay workflow, instrumentation, and data analysis across network laboratories. Approved NCLN assays will be performed at the NCI’s expense, with no need to identify a funding source.

Platform Assay Biomarkers Description
Luminex Immunoassays Apoptosis Multiplex Immunoassays RBM Apoptosis Panel 1: BAX, BAK, Lamin B (Intact and 45 KDa cleaved fragment), SMAC
RBM Apoptosis Panel 2: BAD, BAX: BCL2 heterodimer, BCL-XL, BIM, MCL1
RBM Apoptosis Panel 3: BCL-XL:BAK heterodimer, Active caspase 3, MCl1:BAK heterodimer, Survivin
A novel multiplex immunoassay panel that quantifies 13 biomarkers indicative of the induction, onset, and commitment to apoptosis.
Immunofluorescence Assays γH2AX, pNBS1, and β-Catenin Multiplex Histone H2AX phosphorylated at Ser139, NBS1 phosphorylated at Ser343, β-Catenin This assay detects and quantifies histone H2AX phosphorylated at serine 139 (γH2AX) and NBS1 phosphorylated at serine 343 (pNBS1) using β-Catenin tumor segmentation for pharmacodynamic (PD) evaluation of DNA damage repair status.

SOPs for NCLN-Supported Validated Biomarker Assays

Assay Tissue Specimen Collection Sample Preparation Assay(s) Data Analysis
Apoptosis Multiplex Immunoassays, Luminex tumor SOP340507
Tumor Frozen Needle Biopsy Specimen Collection and Handling
SOP341401
Tumor Biopsy Lysate Fractionation for the Apoptosis Multiplex Immunoassay Panels
SOP341402
Procedure for Use of Bio-Rad Bio-Plex Pro RBM Apoptosis Panel Kits 1-3 for the Analysis of Clinical Tumor Biopsy Extracts and Reporting of Data
γH2AX, pNBS1, and β-Catenin Multiplex, IFA tumor SOP340507
Tumor Frozen Needle Biopsy Specimen Collection and Handling
SOP340550
Tumor Frozen Needle Biopsy Preparation for Pharmacodynamic Immunofluorescence Assays Utilizing Murine Testis and/or Jejunum Control Tissues
SOP340543, γH2AX, pNBS1 IFA Staining with β-Catenin Segmentation for Tumor Biopsy Slides

SOP340544, Whole Slide Image Capture of Tumor Biopsy Slides for γH2AX, pNBS1 Immunofluorescence Assay with β-Catenin Segmentation
SOP340545
Image Extraction and Analysis of Tumor Biopsy Slides from γH2AX, pNBS1 IFA with β-Catenin Segmentation

Additional Supported Validated Biomarker Assays: Overview

DCTD provides training and key reagent support to the cancer research community for the following list of additional supported validated biomarker assays.These assays have been developed employing quality-controlled reagents, standards, and controls. Assay and supporting specimen handling SOPs have been developed to ensure inter-operator, inter-run, and inter-site reproducibility. Rigorous methodologies and reference materials result in accurate and reproducible evaluation of drug effect in heterogeneous clinical specimens.

Platform Assay Biomarkers Description
ELISA Immunoassays MET Immunoassays Intact MET, Dual Phospho-Y1234/Y1235 MET, Phospho-Y1356 MET The MET Immunoassays have been developed to measure the effect of anticancer agents on the levels of intact, dual phospho-Y1234/Y1235, and phospho-Y1356 MET in tumor biopsy samples.
Poly(ADP-Ribose) Poly(ADP-Ribose) This method quantifies poly(ADP-ribose) (PAR) levels as a pharmacodynamic (PD) measurement for PAR polymerase (PARP) inhibitors and/or chemotherapeutic agents.
Immunofluorescence Assays Epithelial-Mesenchymal Transition (EMT) Panel E-cadherin, Vimentin, β-Catenin This assay detects and quantifies the epithelial marker E-cadherin and the mesenchymal marker Vimentin using β-Catenin tumor segmentation for pharmacodynamic (PD) evaluation of epithelial-to-mesenchymal transition.

SOPs for Additional Supported Validated Biomarker Assays

Assay Tissue Specimen Collection Sample Preparation Assay(s) Data Analysis
MET and pMET, ELISA tumor SOP340507
Tumor Frozen Needle Biopsy Specimen Collection and Handling
SOP341201
Biopsy Specimen Processing for MET Immunoassay
SOP341203, Intact MET Immunoassay
SOP341206, Dual Phospho-Y1234/1235 MET Immunoassay
SOP341205, Phospho-Y1356 MET Immunoassay
SOP341208
MET Immunoassay Quality Control, Data Analysis, and Reporting
Poly(ADP-Ribose), ELISA tumor SOP340507
Tumor Frozen Needle Biopsy Specimen Collection and Handling
SOP340520
Biopsy Specimen Processing for PAR Immunoassay
SOP340505
Poly(ADP-ribose) (PAR) Immunoassay
SOP340530
PAR Immunoassay Quality Control, Data Analyses, and Reporting
PBMCs SOP340503
PBMC Specimen Collection, Preparation, and Freezing for Protein Extraction
SOP340506
PBMC Protein Extraction for PAR Immunoassay
Epithelial-Mesenchymal Transition (EMT) Panel, IFA tumor SOP340507
Tumor Frozen Needle Biopsy Specimen Collection and Handling
SOP340550
Tumor Frozen Needle Biopsy Preparation for Pharmacodynamic Immunofluorescence Assays Utilizing Murine Testis and/or Jejunum Control Tissues
SOP340546
EMT Panel IFA Staining for Tumor Biopsy Slides

SOP340547
Whole Slide Image Capture of Tumor Biopsy Slides of EMT Panel IFA
SOP340548
Image Extraction and Analysis of Tumor Biopsy Slides from EMT Panel IFA

Historical Biomarker Assays: Overview

Historical biomarker assays developed by the DCTD Pharmacodynamic Biomarkers Program. Unlike the validated biomarker assays listed above, these additional published assays do not have a formal training program or qualified reagent supply; however, consultation may be requested.

Platform Assay Biomarkers Description
Immunofluorescence Assay (CellSearch System) Immunofluorescence Assay for Circulating Tumor Cells Any biomarker of interest with a qualified antibody conjugate For immunomagnetic selection of circulating tumor cells (CTCs) and immunofluorescent identification of CTCs positive for pharmacodynamic (PD) biomarkers, nuclear or cytoplasmic, to assess the PD effects of anticancer agents.
Immunoassays (ELISA) γH2AX and Total H2AX Immunoassay Histone H2AX phosphorylated at Ser139 and Total H2AX Normalizing γH2AX to the total amount of H2AX provides a more relevant pharmacodynamic readout for monitoring treatment-induced DNA damage.
HIF-1alpha Immunoassay Total HIF-1 alpha HIF1-alpha level is used as a metric for hypoxia in needle tumor biopsies.
Topoisomerase I Immunoassay Total Topoisomerase I This assay measures the effect of TOP1 inhibitors on TOP1 levels in a variety of biospecimen types, including peripheral blood mononuclear cells (PBMCs) and tumor biopsies.

SOPs for Historical Biomarker Assays

Assay Tissue Specimen Collection Sample Preparation Assay(s) Data Analysis
CTC enumeration and γH2AX, CellSearch CTCs LHTP003.08.03
CellSave Tube: Human Blood Collection and Specimen Submission
LHTP003.8.1.1
γH2AX Immunofluorescence Assay Antibody Qualification and Laboratory Proficiency Testing
LHTP003.8.1
Immunofluorescence Assay for Circulating Tumor Cells Using the CellSearch System
HIF-1α, ELISA tumor SOP340902
HIF-1 alpha Immunoassay Tumor Frozen Needle Biopsy Specimen Collection and Handling
SOP340910
Biopsy Protein Extraction for the HIF-1 alpha Immunoassay
SOP340903
HIF-1alpha Immunoassay
SOP340904
HIF-1 alpha Immunoassay Quality Control, Data Analyses, and Reporting
Total Topoisomerase I, ELISA tumor SOP340507
Tumor Frozen Needle Biopsy Specimen Collection and Handling
SOP340702
Biopsy Specimen Processing for TOP1 Immunoassay
SOP340701
Topoisomerase I Immunoassay
SOP340704
TOP1 Immunoassay Quality Control, Data Analyses, and Reporting
PBMCs SOP340503
PBMC Specimen Collection, Preparation, and Freezing for Protein Extraction
SOP340703
PBMC Protein Extraction for TOP1 Immunoassay
SOP340701
Topoisomerase I Immunoassay
SOP340704
TOP1 Immunoassay Quality Control, Data Analyses, and Reporting

SOPs for Specimen Collection

The following specimen collection SOPs were developed by DCTD to standardize the methods for collecting and handling frozen needle biopsies and blood specimens for use in measurement of pharmacodynamic (PD) markers following treatment with anticancer agents.

SOP Description
SOP340507
Tumor Frozen Needle Biopsy Specimen Collection and Handling
Collect and freeze tumor needle biopsies for use in biomarker assays
LHTP003.08.19
Streck Tube: Human Blood Collection and Specimen Submission
Collection, preparation, and shipment of 10 mL human blood specimens in Streck blood collection tubes for use in enrichment and analysis of circulating tumor cells

Please email any questions or comments to PDinfo@mail.nih.gov.