Daniel C. Liebler

Supplementary Figure 1 from Quantification of Antibody–Drug Conjugate Targets in Head and Neck Squamous Cell Carcinoma

Supplementary Figure 2 from Quantification of Antibody–Drug Conjugate Targets in Head and Neck Squamous Cell Carcinoma

Antibody–drug conjugates (ADC) have shown clinical benefit in common solid tumors like breast cancer, lung cancer, and urothelial cancers but are just beginning to be investigated in head and neck cancer. We developed a quantitative immunofluorescence assay to reproducibly and accurately measure HER3 protein concentrations in formalin-fixed, paraffin-embedded tissues. Using this assay, we measured HER3 protein concentrations in two head and neck squamous cell carcinoma (HNSCC) tissue microarray cohorts containing 329 patients. Cell lines with mass spectrometry–measured protein concentrations were used to convert fluorescence signals to protein concentrations in amol/mm2. HER3, EGFR, and TROP2 showed a broad dynamic range in our cohorts with almost all of the cases above our assay’s limit of detection (LOD). Specifically, 100%, 97.5%, and 98.4% of cases have HER3, EGFR, and TROP2 levels above assay LOD. Interestingly, despite the previous demonstration of extremely low HER2 expression in HNSCC, 39.1% of cases showed HER2 expression above our assay LOD of 52.5 amol/mm2. When comparing between therapeutic targets, 97.2%, 99.3%, and 98.6% of cases are double-positive for HER3/EGFR, HER3/TROP2, and EGFR/TROP2, whereas only 34.2%, 34.6%, and 30.5% of cases are double-positive for HER3/HER2, EGFR/HER2, and TROP2/HER2. HER3, EGFR, and TROP2 are highly expressed in HNSCC. HER2 expression is relatively low in this HNSCC cohort compared with other ADC targets, but there are 39.1% of cases with detectable HER2 exceeding historic estimates. Overall, ADC target levels in HNSCC for approved ADCs suggest that patients with HNSCC may benefit from this class of drugs.

Supplementary Figure 3 from Quantification of Antibody–Drug Conjugate Targets in Head and Neck Squamous Cell Carcinoma

Supplementary Figure 5 from Quantification of Antibody–Drug Conjugate Targets in Head and Neck Squamous Cell Carcinoma

Supplementary Figure 6 from Quantification of Antibody–Drug Conjugate Targets in Head and Neck Squamous Cell Carcinoma

Supplementary Figure 7 from Quantification of Antibody–Drug Conjugate Targets in Head and Neck Squamous Cell Carcinoma

Supplementary Figure 4 from Quantification of Antibody–Drug Conjugate Targets in Head and Neck Squamous Cell Carcinoma

Antibody–drug conjugates (ADC) have shown clinical benefit in common solid tumors like breast cancer, lung cancer, and urothelial cancers but are just beginning to be investigated in head and neck cancer. We developed a quantitative immunofluorescence assay to reproducibly and accurately measure HER3 protein concentrations in formalin-fixed, paraffin-embedded tissues. Using this assay, we measured HER3 protein concentrations in two head and neck squamous cell carcinoma (HNSCC) tissue microarray cohorts containing 329 patients. Cell lines with mass spectrometry–measured protein concentrations were used to convert fluorescence signals to protein concentrations in amol/mm2. HER3, EGFR, and TROP2 showed a broad dynamic range in our cohorts with almost all of the cases above our assay’s limit of detection (LOD). Specifically, 100%, 97.5%, and 98.4% of cases have HER3, EGFR, and TROP2 levels above assay LOD. Interestingly, despite the previous demonstration of extremely low HER2 expression in HNSCC, 39.1% of cases showed HER2 expression above our assay LOD of 52.5 amol/mm2. When comparing between therapeutic targets, 97.2%, 99.3%, and 98.6% of cases are double-positive for HER3/EGFR, HER3/TROP2, and EGFR/TROP2, whereas only 34.2%, 34.6%, and 30.5% of cases are double-positive for HER3/HER2, EGFR/HER2, and TROP2/HER2. HER3, EGFR, and TROP2 are highly expressed in HNSCC. HER2 expression is relatively low in this HNSCC cohort compared with other ADC targets, but there are 39.1% of cases with detectable HER2 exceeding historic estimates. Overall, ADC target levels in HNSCC for approved ADCs suggest that patients with HNSCC may benefit from this class of drugs.