Abstract 3366: Progress on development of multiplex panel of 15 biomarkers to support development of anticancer drugs targeting apoptosis.
Authors: Srivastava Apurva K. , Jaganathan Soumya , Stephen Laurie L. , Hollingshead Melinda G. , Scull Jason , Kinders Robert J. , Damour Eric , Donohue Jennifer , Layhee Adam , Mapes James , Esposito Dominic , Tomaszewski Joseph E. , Parchment Ralph E. , Doroshow James H. 
Published: August 2014

Background: The Division of Cancer Treatment and Diagnostics/National Cancer Institute has embarked on development of a multiplex panel of biomarkers of the apoptosis pathway to support proof-of-mechanism studies of investigational agents targeting apoptosis. The selected biomarkers provide quantitative estimate of the commitment, onset, and induction of apoptosis, especially the intrinsic apoptosis pathway. This study updates progress in development of the multiplex panel.

Methods: The immunoassays are built on the Luminex® xMAP multiplex technology platform using magnetic bead capture. The current panel included caspase-3, Lamin-B, Survivin, and markers involved in mitochondrial apoptosis including oligomeric forms of BCL2 family proteins. Calibrators for oligomeric proteins, which are formed in vivo by the non-covalent association of BH3 domain proteins, are developed as synthetic recombinant fusion proteins. The panel is designed for analysis of biomarkers in fractionated cell lysates prepared from flash frozen tumor biopsies. Fit-for-purpose approach is utilized for validation of biomarkers.

Results: We have completed development of 15 biomarkers in the panel. Highly specific and sensitive sandwich immunoassays were designed using capture & labeled antibody pairs and recombinant proteins as calibrators. To overcome complications arising from the multiple readout of some markers (due to protein-protein interactions), the multiplex is grouped in three smaller panels: Panel 1(BAK, BAX, Total Caspase-3, Lamin B(intact +45kDa), & SMAC); Panel 2 (BAD, BAX-BCL-2, BCL-xl, BIM& Mcl-1); and Panel 3 (Active Caspase-3, BCL-xl - BAK, MCL-1 - BAK, phospho-BAD, & Survivin). Specificity of oligomeric forms of biomarkers, BAK-MCL1, BAX-Bcl2, BAK-BCL-xl, and BAX-BAK was achieved by capturing BAK and BAX protein and probing with labeled antibodies specific to MCL-1, BCL-xl, BAK, or BAX. Lysates from tumor biopsy samples can be examined in as little as 50 μg of protein. Specificity of the oligomeric assays was also confirmed by immunoprecipitation and immunoblotting techniques. Fit-for-purpose validation studies are ongoing.

Conclusions: The development phase of the multiplex panel is completed. The panel is now ready for transfer from the development laboratory to clinical laboratories for first-in-human use. In the next phase, the panel will be launched as commercial assay kits. One unique feature of the panel is the ability to measure novel oligomeric biomarkers that mimic alterations in protein-protein interactions. This dynamic readout of intrinsic pathway proteins could provide much needed direct evidence for activity of important classes of molecularly targeted drugs in early clinical trials. Funded by NCI Contract No HHSN261200800001E. First three authors contributed equally to the study.

Citation Format: Apurva K. Srivastava, Soumya Jaganathan, Laurie L. Stephen, Melinda G. Hollingshead, Jason Scull, Robert J. Kinders, Eric Damour, Jennifer Donohue, Adam Layhee, James Mapes, Dominic Esposito, Joseph E. Tomaszewski, Ralph E. Parchment, James H. Doroshow. Progress on development of multiplex panel of 15 biomarkers to support development of anticancer drugs targeting apoptosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3366. doi:10.1158/1538-7445.AM2013-3366