Abstract

We have previously described the ADAPT Biotargeting SystemTM as a novel platform for highly multiplexed poly-ligand profiling of complex phenotypes such as drug response (1). Here we report extended capabilities of the this platform for target identification directly from formalin-fixed-paraffin embedded (FFPE) tissues using aptamer libraries enriched toward HER2+ breast cancer. Standard mass spectrometry-based biomarker and drug target discovery from FFPE tissues can be challenging due to limited amounts of tissue, harsh conditions of fixation and extraction and the general problem of masking by highly abundant proteins. A single stranded-oligodeoxynucleotide aptamer library was enriched on HER2+ FFPE breast cancer specimens and conjugated with biotin as well as a label transfer reagent, Sulfo-NHS-SS-Diazirine (Sulfo-SDAD). The biotinylated-SDAD conjugated library (B-SDAD-EL) was applied to HER2+ FFPE tissues and photocrosslinked to cognate binding partners within the FFPE sample in order to preserve aptamer-protein interactions under harsh denaturing conditions required for protein extraction and sample preparation. Aptamer-protein complexes were affinity purified and the label was transferred from bound aptamers to their binding partners under reducing conditions that enable proteomic digestion and high resolution mass spectrometry detection. An open database search was performed where the precursor ion tolerance was set to ± 500 Da for database searching, which enabled identification of peptides containing the transferred label as well as additional unknown variable modifications induced by the tissue fixation process. We identified proteins with known roles in HER2+ breast cancer along with several potentially drugable targets not previously associated with HER2 positivity. Differential expression of candidate targets was orthogonally confirmed by immunohistochemistry. By nature of its extreme molecular complexity and its ability to be enriched or “trained,” toward phenotypes of interest, the ADAPT Biotargeting SystemTM can be deployed to advance precision medicine by identifying predictive biomarkers and drug targets with novel associations to complex interactomes.

Conclusions

• Other aptamer based selections on FFPE tissue identified targets from cell cultures (4, 5). ADAPT™ enrichment combined with aptamer based affinity labeling (ABAL) (6) provides an unbiased approach for target identification directly from FFPE tissue.
• We identified proteins with known roles in HER2+ breast cancer along with several potential biomarkers not previously associated with HER2 positivity. Additional cases are needed to confirm prognostic value.
• ADAPT™ allows simultaneous detection of multiple targets highlighting potential metabolic pathway differences between two subtypes of breast cancer with a single polyligand approach.
• Advantages of enrichment on tissue slices includes the ability to use clinical specimens. See Poster P2-09-09, highlighting the ability to not only select between two breast cancer subtypes but also drug responder vs. nonresponder cohorts.