Quantification of Her2 expression in Her2-low tumors by targeted mass spectrometry

Introduction

In approximately 15% of breast cancer cases, ERBB2 gene amplification leads to an overexpression of human epidermal growth factor receptor 2 (HER2) protein, resulting in a worse prognosis unless patients receive HER2-targeted therapy. However, roughly half of breast cancer cases exhibit low levels of HER2 expression without ERBB2 amplification. These are termed HER2-low. Unfortunately, these cases are excluded from HER2-targeted therapy. Nevertheless, there is hope with the emergence of new drugs targeting HER2, which have shown promising results in treating HER2-low breast cancers. This underscores the need for more accurate methods of determining HER2 status, given the limitations of current testing techniques such as IHC. To address these challenges, our efforts have been focused on developing a targeted mass spectrometry-based assay for the HER2 peptide, GLQSLPTHDPSPLQR. Our hypothesis is that this approach will provide the necessary assay sensitivity to quantify HER2 in HER2-low tumors, thereby refining patient selection for treatment with HER2-targeting antibody-drug conjugates.

Method

In this ongoing study, we refined and validated a PRM-PASEF assay to measure HER2 levels using clinical FFPE samples from both patient-derived xenografts (PDX) and cancer patients. After standard assessments such as H&E staining and IHC, we used nanoflow liquid chromatography and PRM-mass spectrometry to quantify HER2 in diverse HER2 status samples. The final step involves correlating the HER2 status (based on our assay) with the IHC results, ensuring its accuracy and reliability.

Result

The PRM-PASEF assay, targeting the GLQSLPTHDPSPLQR peptide, was developed for HER2 protein quantification. Characterization through response curves used synthetic peptides for calibration and isotope-labeled standards for quantitation, covering almost 3 orders of magnitude across 5 concentration points. The quantification of endogenous concentrations of the HER2 peptide revealed that the majority of values were above the lower limit of quantification (LLOQ). Moreover, a significant positive correlation was found between normalized HER2 concentrations and their IHC results.

Conclusion

Our targeted assay for HER2 quantification shows potential in improving HER2 status assessment, especially in distinguishing HER2 low cases from negative ones. This could reduce the need for repeat IHC or FISH tests, lowering diagnosis time and costs for breast cancer patients. The simplified assay procedure also can lower the barriers to entry for the clinical application of the targeted assay..