HER2 and TOP2A in high-risk early breast cancer patients treated with adjuvant epirubicin-based dose-dense sequential chemotherapy

Table 1 Proteins, source and dilution of antibodies, staining procedures and patterns and interpretation of results

Protein	Antibody [clone, source]	Ab dilution	Pretreatment/EU	IT	IHC Staining Detection System/Chromogen	Scoring System	Cut-off (%)	Staining Pattern	Reference
Ki67	MIB1 (1)	1:70	15'/EU2	20'	Polymer HRP/DAB	SQ	≥ 14%*	Nuclear	[28]
HER2	PL (1)	1:200	25'/EU1	30'	Polymer HRP/DAB	0-3	> 30%^#	Membranous	[23]
TopoIIa	KiS1 (1)	1:200	15'/EU2	30'	EnVision/DAB	SQ	> 5%**	Nuclear	[27]
ER	6F11 (2)	1:70	20'/EU1	20'	Polymer HRP/DAB	H-Score	≥ 1%	Nuclear	[29]
PgR	1A6 (2)	1:70	20'/EU1	20'	Polymer HRP/DAB	H-Score	≥ 1%	Nuclear	[29]

Antigen Retrieval was done on a hot plate at 98°C.
DAB: 3,3'-Diaminobenzidine; EU: Epitope Unmasking; EU1: Citric acid, pH 6.0; EU2: Ethylenediaminetetraacetate, pH 8.8; HRP: Horseradish Peroxidase; IT: Incubation time; SQ: Semiquantitative.
(1): Dako, Glostrup, Denmark; (2): Leica Biosystems, Newcastle Upon Tyne, UK.
*: Proliferation Index: Low if < 14% and High if ≥14% positive nuclei were observed; #: score 3+ in cases with > 30% positive tumor cells; **: positive if at least moderate staining intensity in > 5% of tumor cells.

ISSN: 1479-5876