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Abstract Introduction:
Human epidermal growth factor receptor 2 (HER2) over-expression is a driver of breast cancer tumorigenesis in a subset of patients. HER2-positive metastatic breast cancer is treated with anti-HER2 antibodies, such as trastuzumab. In the clinic, HER2-positive is defined as immunohistochemistry (IHC) 3+ or IHC 2+ and FISH-positive. Novel anti-HER2 antibody drug conjugates may be clinically effective even with lower levels of HER2 expression necessitating more sensitive and robust quantification of HER2. Mass spectrometric targeted assays have shown several advantages over IHC assays, including absolute quantitation and enhanced multiplexing. To maximize the sensitivity and the selectivity of a clinical MS assay, we have taken advantage of ion mobility separation of peptides, subsequent to reverse-phase nanoLC separation and prior to separation in the gas-phase. We have developed a targeted HER2 mass spectrometric assay utilizing FAIMS separation, coupled to a high resolution Orbitrap detector, and applied it to FFPE tissue tumor biopsies.
Methods:
The HER2 FAIMS-PRM assay utilized an EvoSep One nanoLC system (EvoSep, Odense, DK) coupled to an Orbitrap Fusion Lumos mass spectrometer with a FAIMS-PRO interface (Thermo Scientific, San Jose, CA). Formalin-fixed HeLa cells and human tissue were used as a complex matrix to determine the limit of detection (LOD) and limit of quantitation (LOQ). A ten-point dilution series was acquired in triplicate, ranging from 5 amol to 100 fmol on-column. Each analysis consisted of 1 µg matrix or sample spiked with stable-isotope labeled internal standard peptide (5 fmol; ELVSEFSR).
Test samples included HER2+ and HER2- FFPE breast cancer samples Tumor-rich regions were collected by laser microdissection using LEICA LMD 6500 (LEICA Biosystems, Germany), and digested with trypsin. PRM data was analyzed using Skyline software version 4.2 (University of Washington, WA).
Results:
The optimal compensation voltage for HER2: ELVSEFSR [M+2H]2+ was -57 V. For the ELVSEFSR peptide, LLOQ changed from 137 amol to 45 amol with ion-trap detection, and improved CV was observed with Orbitrap detection. We demonstrated that this assay allows absolute quantitation of HER2 levels in FFPE tumor samples at attomole levels.
Conclusions:
FAIMS-PRM further improves the conventional PRM performance and offers more sensitive biomarker quantitation in the clinical setting, applicable to HER2 quantitation in FFPE tumor biopsies. We see a peptide-specific range of sensitivity improvement with FAIMS, presumably dependent upon the level of interference without FAIMS and the degree to which FAIMS is able to remove the interfering peaks. This technology will allow us to investigate patient stratification for anti-HER2 therapy below the current IHC 2+/3+ thresholds.
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= Discovery stage.
= Translation stage.
= Clinically available.
