DCTD Programs
Last Updated: 03/16/2023
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 | Biomarker Name | Biomarker Assay Details | Description |
|---|---|---|---|
| Luminex Immunoassays | AKT Multiplex Immunoassay (Panels 1-3) ERK/MEK Multiplex Immunoassay (Panels 4-5) |
Panel 1 (Total AKT/rpS6 ): Total AKT1, Total AKT2, Total AKT3, Total rpS6 Panel 2 (Phospho AKT/rpS6 A): pS473-AKT1, pS474-AKT2, pS472-AKT3, pS235-rpS6 Panel 3 (Phospho AKT/rpS6 B): pT308-AKT1, pT309-AKT2, pT305-AKT3, pS240/244-rpS6 Panel 4 (Total ERK/MEK): Total ERK1, Total ERK2, Total MEK1, Total MEK2 Panel 5 (Phospho ERK/MEK): pT202/Y204-ERK1, pT185/Y187-ERK2, pS218/222-MEK1, pS222/226-MEK2 |
Novel isoform-specific signaling multiplex immunoassay panels of 12 biomarkers for AKT and rpS6 and 8 biomarkers for MEK and ERK to measure on-target or downstream pathway modulation by multiple classes of drugs including PI3K/AKT/mTORC and RAS/RAF/MEK/ERK. |
| Apoptosis Multiplex Immunoassay | 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. | |
| Multiplex Immunofluorescence Assays | γH2AX, pNBS1 IFA with β-CATN Segmentation | 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
| Biomarker Name | Tissue | Specimen Collection | Sample Preparation | Assay(s) | Data Analysis |
|---|---|---|---|---|---|
| AKT Multiplex Immunoassay (Panels 1-3), Luminex ERK/MEK Multiplex Immunoassay (Panels 4-5), Luminex |
tumor | SOP340507 Tumor Frozen Needle Biopsy Specimen Collection, Handling and Shipping for PADIS, Frederick National Laboratory for Cancer Research (FNLCR) SOP340567 Tumor Frozen Needle Biopsy Specimen Collection, Handling and Shipment to EET Biobank |
SOP341210 Whole Cell Lysate Biopsy Specimen Processing for Immunoassays |
SOP341404 Procedure for use of Signaling Multiplex Panels 1-3 (AKT/rpS6) for the Analysis of Clinical Tumor Biopsy Extracts and Reporting of Data SOP341405 Procedure for use of Signaling Multiplex Panels 4-5 (MEK/ERK) for the Analysis of Clinical Tumor Biopsy Extracts and Reporting of Data |
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| Apoptosis Multiplex Immunoassay, Luminex | tumor | SOP340507 Tumor Frozen Needle Biopsy Specimen Collection, Handling and Shipping for PADIS, Frederick National Laboratory for Cancer Research (FNLCR) SOP340567 Tumor Frozen Needle Biopsy Specimen Collection, Handling and Shipment to EET Biobank |
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 |
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| γH2AX, pNBS1 IFA with β-CATN Segmentation, IFA | tumor | SOP340507 Tumor Frozen Needle Biopsy Specimen Collection, Handling and Shipping for PADIS, Frederick National Laboratory for Cancer Research (FNLCR) SOP340567 Tumor Frozen Needle Biopsy Specimen Collection, Handling and Shipment to EET Biobank |
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 |
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| 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, Handling and Shipping for PADIS, Frederick National Laboratory for Cancer Research (FNLCR) |
Collect and freeze tumor needle biopsies for use in biomarker assays and preparation for shipment to PADIS, FNLCR |
| SOP340567 Tumor Frozen Needle Biopsy Specimen Collection, Handling and Shipment to EET Biobank |
Collect and freeze tumor needle biopsies for use in biomarker assays and preparation for shipment to EET Biobank |
| 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.