Background: Conventional HER2-targeting therapies improve outcomes for patients with HER2-positive breast cancer (BC), defined as tumors showing HER2 protein overexpression by an immunohistochemistry (IHC) assay and/or ERBB2 gene amplification determined by in situ hybridization (ISH). Emerging HER2-targeting compounds show benefit in some patients with neither HER2 protein overexpression nor ERBB2 gene amplification, creating a need for new assays to select HER2-low tumors for treatment with these compounds. Here, we report the analytical performance of a targeted mass spectrometry-based assay for quantifying HER2 protein in formalin fixed paraffin-embedded (FFPE) and frozen BC biopsies. Methods: We used immuno-enrichment coupled to multiple reaction monitoring-mass spectrometry (immuno-MRM-MS) to quantify HER2 protein (as peptide GLQSLPTHDPSPLQR) in 96 frozen and 119 FFPE BC biopsies. We characterized linearity, lower limit of quantification (LLOQ), and intra- and inter-day variation of the assay in frozen and FFPE tissue matrices. We determined concordance between HER2 immuno-MRM and predicate IHC and ISH assays and examined the benefit of multiplexing the assay to include proteins expressed in tumor sub-compartments (e.g. stroma, adipose, lymphocytes, epithelium) to account for tissue heterogeneity. Results: HER2 immuno-MRM assay linearity was ≥103, assay coefficient of variation (CV) was 7.8% (FFPE) and 5.9% (frozen), and endogenous measurement CV was 7.7% (FFPE) and 7.9% (frozen). Immuno-MRM-based HER2 measurements strongly correlated with predicate assay HER2 determinations, and concordance was improved by normalizing to GAPDH. HER2 was quantified above LLOQ in all HER2-low and -negative tumors. Conclusions: Immuno-MRM-MS can be used to quantify HER2 in FFPE and frozen BC biopsies, even at low HER2 expression levels.

Slide-mounted FFPE tissue sections were deparaffinized, rehydrated, and protein was extracted into a 0.1% RapiGest solution. The frozen tissue was cryopulverized and the protein was extracted into a 6M Urea solution. Protein concentration was quantified by Micro BCA Assay (ThermoFisher). Up to 50 µg protein from FFPE samples and 100 µg from frozen samples were reduced and alkylationalkylated, then incubated at 37 °C for two hours after trypsin addition at 1:50 trypsin:protein ratio by mass and then overnight at 37 °C after two additions of trypsin were addedaddition at 1:50 and 1:100 trypsin:protein ratio by mass. After digestion, a mixtumre of SIS peptides was spiked into the individual samples at 80 fmol/sample of protein lysate, and the samples were desalted. Peptide immunoaffinity enrichment fonsisted of overnight incubations of the cross-linked mAbs using a mix of at least 1 µg of each of the 23 mAbs. A KingFisher platform was used to wash the beads. For LC-MRM-MS analysis, ten 10 µL of a 26 µL eluate were injected on an ekspert nanoLC 425 with an NLC 400x AS autosampler coupled to a 6500 QTRAP mass spectrometer.