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- Quantitative Protein Expression of Antibody-Drug Conjugate Targets in EGFR Mutated and Wild-Type Non-Small Cell Lung Cancer

# Quantitative Protein Expression of Antibody-Drug Conjugate Targets in EGFR Mutated and Wild-Type Non-Small Cell Lung Cancer

Trontzas IP, He M, Wurtz A, Robbins CJ, Robinson N, Bates K, et al.

Clinical Cancer Research . 2025;31(13):2767-2776. DOI [10.1158/1078-0432.CCR-24-3347](https://doi.org/10.1158/1078-0432.CCR-24-3347). PMID 40047548. PMCID PMC12213210.

How to cite

### AMA

Trontzas IP, He M, Wurtz A, Robbins CJ, Robinson N, Bates K, et al. Quantitative Protein Expression of Antibody-Drug Conjugate Targets in EGFR Mutated and Wild-Type Non-Small Cell Lung Cancer. Clin Cancer Res . 2025;31(13):2767-2776. doi:10.1158/1078-0432.CCR-24-3347

### APA

Trontzas, I. P., He, M., Wurtz, A., Robbins, C. J., Robinson, N., Bates, K., et al. (2025). Quantitative Protein Expression of Antibody-Drug Conjugate Targets in EGFR Mutated and Wild-Type Non-Small Cell Lung Cancer. Clinical Cancer Research , 31(13), 2767-2776. https://doi.org/10.1158/1078-0432.CCR-24-3347

### BibTeX

@article{trontzas2025quantitative,
title = {Quantitative Protein Expression of Antibody-Drug Conjugate Targets in EGFR Mutated and Wild-Type Non-Small Cell Lung Cancer},
author = {Trontzas, I. P. and He, M. and Wurtz, A. and Robbins, C. J. and Robinson, N. and Bates, K. and others},
journal = {Clinical Cancer Research},
year = {2025},
volume = {31},
number = {13},
pages = {2767--2776},
doi = {10.1158/1078-0432.CCR-24-3347},
pmid = {40047548}
}

Antibody-drug conjugates are reshaping treatment for non-small cell lung cancer, but only a fraction of patients benefit, and the tests that pick them are the weak link. Target level is usually read by a pathologist scoring a stained slide by eye, a semi-quantitative call that separates the low range poorly and can miss patients who could still respond.

This Yale team measured four ADC target proteins, HER2, TROP2, HER3 and EGFR, as absolute concentrations using mass-spectrometry-calibrated quantitative immunofluorescence across three NSCLC tissue-microarray cohorts split by EGFR mutation status. All four targets were broadly expressed in every cohort, with high proportions of cases above assay limits, arguing that ADC benefit may reach well beyond mutation-selected patients and that objective quantitation could sharpen who gets treated.

[Read publication at Clinical Cancer Research](https://pmc.ncbi.nlm.nih.gov/articles/PMC12213210/)

## Key findings

- All four ADC targets showed broad, comparable expression across the three NSCLC cohorts. Above the assay limit of detection, HER2 reached 100%, 100% and 99.6% of cases and EGFR 100%, 100% and 98.5% in the EGFRmut, EGFRwt and unknown-status cohorts.

- Each target tracked EGFR biology in its own way. HER2 expression was significantly associated with EGFR expression (p=0.0005) but not with EGFR mutation (p=0.14), HER3 correlated negatively with EGFR mutation (p

## CyteFinder in the methods

“Fluorescent images of HER2-TROP2 and EGFR-stained TMAs were acquired using the CyteFinder HT imaging system (RareCyte Inc., Seattle, WA, USA). Acquired images were analyzed in QuPath (v.4.3.0), an open-source image analysis software for digital pathology, and targets were quantified using Qymia, an in-house developed QuPath plugin for automated QIF/IHC analysis.”

— Trontzas et al., Clinical Cancer Research (2025), Methods, “Image acquisition and analysis”

## Why it matters for CyteFinder users

If you are weighing CyteFinder for quantitative tissue imaging, look at the role it played here. Turning a stained slide into an absolute protein concentration only works if the raw fluorescent signal is captured the same way on every core, in every batch, on every day; if it is not, the calibration curve that converts intensity to amol/mm2 stops meaning anything. That capture is the job the instrument did. The HER2-TROP2 multiplex and the EGFR singleplex assays were imaged on CyteFinder across all three tissue-microarray cohorts, then handed to QuPath and the Qymia plugin, which assigned each target to a molecular compartment and scaled exposure-normalized intensities to absolute abundance using mass-spectrometry-anchored cell-line standards. For your own workflow, the point is that quantitation is won or lost at acquisition: a standardized, multi-channel capture is what lets downstream analysis anchor readings to a standard curve and report a target as a comparable, absolute number rather than a subjective score.

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