The ability to accurately quantify proteins in formalin-fixed paraffin-embedded tissues using targeted mass spectrometry opens exciting perspectives for biomarker discovery as it would provide access to a wealth of clinically annotated samples for multiplexed protein quantification. We have developed and evaluated a selected reaction monitoring (SRM) assay for the human receptor tyrosine-protein kinase erbB-2 (HER2) in formalin-fixed paraffin-embedded breast tumors. Peptide candidates were identified using an untargeted mass spectrometry approach in relevant cell lines. A multiplexed assay was developed for the six best candidate peptides and evaluated for linearity, precision and lower limit of quantification. Results showed a linear response over a calibration range of 0.012 to 100 fmol on column (R2: 0.99–1.00).The lower limit of quantification was 0.155 fmol on column for all peptides evaluated. The six HER2 peptides were quantified by selected reaction monitoring in a cohort of 40 archival formalin-fixed paraffin-embedded tumor tissues from women with invasive breast carcinomas, showing different levels of HER2 gene amplification. The amounts of the six HER2 peptides were highly and significantly correlated with each other, indicating that peptide levels can be used as surrogates of protein amounts in formalin-fixed paraffin-embedded tissues. After normalization for tissue sample size, selected reaction monitoring peptide measurements were able to correctly predict 90% of cases based on HER2 amplification as defined by the American Society of Clinical Oncology and College of American Pathologists. In conclusion, the developed assay showed good analytical performances and a high agreement with immunohistochemistry and fluorescence in situ hybridization data. This study demonstrated that selected reaction monitoring allows to accurately quantify protein expression in formalin-fixed paraffin-embedded tissues and represents therefore a powerful approach for biomarker discovery studies.

Suitable HER2 peptides for targeted MS analysis were identified by untargeted MS analysis from untreated and formalin-fixed HER2-overexpressing cell lines (SKBR-3 and BT-474) (data available via ProteomeXchange). The list of peptide sequences observed using the untargeted MS approach and suitable for developing an SRM assay (proteotypic and containing no methionine) were refined using Skyline v1.3 software (MacCoss Lab, Seattle, WA, USA) and an SRM assay was developed and evaluated for the six best performing peptides. For quantification, external calibration curves were built by spiking light and heavy-labeled AQUA peptides in a complex non-human protein background (bacterial protein extract). The peptides of interest were quantified in a cohort of 40 archival formalin-fixed paraffin-embedded tumor tissues from women with invasive breast carcinomas. The samples showed different levels of HER2 gene amplification as assessed by standard methods used in clinical pathology. After normalization for sample size, the agreement between peptide levels measured by SRM and HER2 amplification as defined by the American Society for Clinical Oncology was tested using ordinal logistic regression. Several normalization strategies were evaluated to account for the size of the sample.

Formalin-fixed paraffin-embedded (FFPE) breast tumor resection samples were selected retrospectively from the archive of the Division of Clinical Pathology of the Geneva University Hospitals from 2001 to 2011 (the study was approved by the ethical committee of the Canton of Geneva (protocol NAC 13-109)). Samples included cases of tumorectomy or mastectomy from neo-adjuvant treatment-naïve women with invasive breast carcinomas, including 34 ductal, 3 lobular, 1 micropapillary, 1 mucinous and 1 medullary. Exclusion criteria were male gender and administration of neo-adjuvant treatment. For all cases, HER2 amplification was assessed by fluorescence in situ hybridization (FISH) as part of the routine procedure in the Division of Clinical Pathology. The cohort included 40 patients distributed in 4 groups of 10 patients each, according to their HER2 amplification status as measured by FISH: 10 patients with no HER2 amplification (FISH ratio <2, all ductal) and 3 groups of 10 patients, each with increasing HER2 gene amplification level, as determined by a FISH ratio of 2-4 (9 ductal and 1 medullary); 4-10 (9 ductal and 1 lobular); and >10 (6 ductal, 2 lobular, 1 mucinous, 1 micropapillary), respectively. In addition, HER2 overexpression was assessed by IHC for all cases. HER2 FISH and IHC scoring were performed according to the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) recommendations and two expert breast pathologists scored independently the IHC and FISH analyses under a microscope with no digital quantification. 20 µm thick tissue slices cut using a microtome were mounted on glass slides for dissection and protein extraction. Only the infiltrative tumor area, omitting ductal or lobular in situ carcinoma was delimited by a pathologist expert in breast pathology on a separate tissue slice stained with hematoxylin and eosin (H&E). Scalpel macrodissection of the tumor was performed by superimposing each 20 µm slice with the H&E template. The dissected areas were deparaffinated and rehydrated, after which the tissue was collected and stored at -80 °C until protein extraction. The collected tissue recovered from a 20 µm thick slice was heated at 100°C in presence of RapiGest™ SF and 1,4-dithioerythritol (DTE). Following sonication, samples were heated again at 80 °C for 2 hours, after which they underwent a second round of sonication followed by alkylation and tryptic digestion. The samples were desalted and the dry extract was resuspended in mobile phase for LC-MS analysis.