The Breast Cancer Proteome and Precision Oncology

Abstract

The goal of precision oncology is to translate the molecular features of cancer into predictive and prognostic tests that can be used to individualize treatment leading to improved outcomes and decreased toxicity. Success for this strategy in breast cancer is exemplified by efficacy of trastuzumab in tumors overexpressing ERBB2 and endocrine therapy for tumors that are estrogen receptor positive. However, other effective treatments, including chemotherapy, immune checkpoint inhibitors, and CDK4/6 inhibitors are not associated with strong predictive biomarkers. Proteomics promises another tier of information that, when added to genomic and transcriptomic features (proteogenomics), may create new opportunities to improve both treatment precision and therapeutic hypotheses. Here, we review both mass spectrometry-based and antibody-dependent proteomics as complementary approaches. We highlight how these methods have contributed toward a more complete understanding of breast cancer and describe the potential to guide diagnosis and treatment more accurately.

Figure 1.

Overview of mass-spectrometry (MS)-based approaches. (A) For discovery MS-based methods, extracted proteins are subjected to enzymatic digestion to generate peptides, which are subsequently analyzed by liquid chromatography/tandem MS. Downstream data-dependent acquisition (DDA) selects a predefined number of MS1 precursor ions for fragmentation. In contrast, data-independent acquisition (DIA) scans all MS1 precursor ions and selects a window containing ions for fragmentation in MS2. (B) For targeted MS-based methods, predetermined peptides are quantified and heavy-isotope-labeled peptides can be spiked-in as internal standards for precise quantification. Multiple reaction monitoring (MRM), single-reaction monitoring (SRM), and parallel reaction monitoring (PRM) acquisition methods can be used to capture and measure specific peptides of interest and allows for quantification of low-abundance peptides.

Figure 2.

Antibody-based proteomic approaches. In forward-phased arrays, antibodies or aptamers are bound to a solid-phase platform, usually a glass slide or beads. The array is incubated with a protein extract or with plasma/serum to measure up to 7,000 proteins simultaneously in a single sample. In reverse-phase arrays, protein lysates from up to 1,000 samples are printed onto a solid phase such as glass slides or nitrocellulose membranes. The arrays are then incubated with a single antibody in optimized conditions. Up to 500 antibodies can be measured in a single experiment by staining replicate arrays. As an example of spatially resolved single-cell proteomics in the CODEX platform, slides with the tissue of interest are incubated with a DNA-conjugated antibody solution, which can contain up to 60 different antibodies. Antibody binding is then revealed through multiple cycles of hybridization with fluorescent-labeled complementary DNA, imaging, and removal of the fluorescent DNA. Image processing then allows alignment of images on the tissue slide with segmentation of individual cells facilitating spatial single-cell analysis. Other spatially oriented approaches such as cyclic-immunofluorescence (cyc-IF) and multiplex IHC (mIHC) use a variation of the approach encompassing the concept of staining, imaging, and erasing signals through multiple cycles.