Bridging practice and precision: a quantitative HER2 protein assay ready for clinical use in guiding trastuzumab deruxtecan therapy

Purpose Current daily usage of Trastuzumab Deruxtecan (T-DXd) is guided by immunohistochemistry (IHC)-based HER2 assessment, with known inconsistency and inaccuracy to differentiating IHC 0 from 1 +. In this study, a quantitative HER2 assay based on the Quantitative Dot Blot (QDB) method was explored to fill this unmet need. Methods Consecutive resection specimens of HER2 IHC 0 and 1+ from invasive breast cancer patients were assigned to training (n=106) and validation cohorts ( n= 119), respectively by admission time. Protein lysates were extracted from 2x5 μm FFPE slices for HER2 quantification while the adjacent slice was used for IHC staining. Results QDB was demonstrated to be more consistent than IHC with an inter-rater Intraclass Correlation Coefficient (ICC) of 0.877 (95%CI: 0.840-0.908) vs. 0.513 (95%CI: 0.433-0.601). Receiver Operating Characteristic (ROC) analysis was performed benchmarked with unanimous agreement of 18 pathologists in the training cohort to achieve an Area Under the Curve (AUC) of 0.9477 (p0.005). A cutoff of 0.2746 nmole/g was also identified with its imprecision interval to stratify specimens into 0 (C 5 ), equivocal (≥ C 5 ≤ C 95 ) and 1+ ( C 95 ), with overall concordance of 90.9% and 87.3% when benchmarked with unanimous and consensus agreement (≥75%) of pathologists in the training cohort, and 92.0% and 90.5% when validated double-blinded with 12 pathologists in the validation cohort. More importantly, even among specimens unanimously categorized as IHC 0, there were ~15% specimens classified as 1+ by QDB. Conclusion Our results supports the QDB HER2 assay as an alternative option to guide T-DXd daily usage by distinguish Her2-low from Her2 0 while setting the stage for outcome-based patient stratification.