Analytical Validation of a Highly Quantitative, Sensitive, Accurate, and Reproducible Assay (HERmark^®) for the Measurement of HER2 Total Protein and HER2 Homodimers in FFPE Breast Cancer Tumor Specimens

Table 1

Accuracy of cell line controls as measured by flow cytometry, IHC, and ELISA. The majority of the cell lines used for accuracy studies are of breast origin with the exception of MDA-MB-435 which has been shown to be of melanoma origin and A431, which is from an epidermoid carcinoma of the vulva. All cell lines were grown in-house and FFPE blocks prepared on the same day as lysates prepared for subsequent HER2 ELISA assay. The flow cytometry was performed on the same day that the cells were fixed and made into blocks. The ELISA experiments represent an 𝑛=3 experiments performed on separate days. The flow cytometry assays were done in triplicate on the same as described in materials and methods.

Cell Line	HER2 IHC Score	Flow Cytometry HER2 (receptors/cell)	Flow Cytometry %CV	ELISA [HER2] ng/mg	ELISA %CV
BT474	3+	499795.2	10.4	295.5	15.9
MDA_MB_361	2/3+	212456.0	1.9	65.7	7.4
BT-20	N/A	64,386	3.6	15.5	13.0
ZR-75-1	1+	63,708	2.2	22.1	6.4
T47D	0/1+	32303.1	8.2	16.3	11.3
A431	0	27343.1	0.8	23.3	2.3
MDA_MB_231	0	11249.6	21.5	5.1	43.7
MDA_MB_435	0	2420.5	6.1	2.9	70.7
MDA_MB_468	0	10567.1	17.0	1.0	na