References

Antibodies and Methods Used in Immunohistochemical Analysis of Cell Lines

Study Code Antibody and Supplier* Dilution Incubation Time (min)^† Antigen Retrieval System Antigen Retrieval Buffer Detection Automation
A Novocastra clone CB11 1:400 40 PC, 2.5 min 10-mmol/L EDTA, pH 7.0 DAKO Envision Plus Life Sciences Sequenza
B Novocastra clone CB11 1:150 60 PC, 2 min 10-mmol/L citrate, pH 6.0 Vector Elite ABC None
C Novocastra clone CB11 1:160 45 None -- Vector Elite ABC A. Menarini Optimax
D Novocastra clone 10A7 1:20 45 None -- Vector Elite ABC A. Menarini Optimax
E DAKO HercepTest^‡ Life Sciences Sequenza
F DAKO polyclonal A0485 1:1,500 25 95°C water bath, 45 min 5-mmol/L EDTA, pH 7 Labeled Streptomyces avidin DAKO Techmate 500
G Oncogene Research Products clone 3B5 1:1,500 45 PC, 2 min 10-mmol/L citrate, pH 6.0 Streptomyces ABC None
ABC, avidin-biotin complex; PC, pressure cooker pretreatment.
* DAKO, Ely, Cambridgeshire, England; Life Sciences International, Basingstoke, Hampshire, England; Menarini Diagnostics, Finchampstead, Berkshire, England; Novocastra Laboratories, Newcastle upon Tyne, England; Oncogene Research Products, CN Biosciences UK, Nottingham, England; Vector Laboratories, Peterborough, England.
† Performed at room temperature unless stated otherwise.
‡ All procedures performed as directed in the HercepTest kit.

Study Code	Antibody and Supplier*	Dilution	Incubation Time (min)^†	Antigen Retrieval System	Antigen Retrieval Buffer	Detection	Automation
A	Novocastra clone CB11	1:400	40	PC, 2.5 min	10-mmol/L EDTA, pH 7.0	DAKO Envision Plus	Life Sciences Sequenza
B	Novocastra clone CB11	1:150	60	PC, 2 min	10-mmol/L citrate, pH 6.0	Vector Elite ABC	None
C	Novocastra clone CB11	1:160	45	None	--	Vector Elite ABC	A. Menarini Optimax
D	Novocastra clone 10A7	1:20	45	None	--	Vector Elite ABC	A. Menarini Optimax
E	DAKO HercepTest^‡						Life Sciences Sequenza
F	DAKO polyclonal A0485	1:1,500	25	95°C water bath, 45 min	5-mmol/L EDTA, pH 7	Labeled Streptomyces avidin	DAKO Techmate 500
G	Oncogene Research Products clone 3B5	1:1,500	45	PC, 2 min	10-mmol/L citrate, pH 6.0	Streptomyces ABC	None

Results of Fluorescence In Situ Hybridization Analysis

Study Center/Cell Line No. of Nuclei Counted Total Signals (Mean per Nucleus) HER-2/CEP 17 Ratio Conclusion
HER-2/neu CEP
University of Wales Hospital, Cardiff
   SKOV-3 59 571 (9.6) 212 (3.6) 2.7 Amplified
   MDA-MB-453 60 504 (8.4) 237 (4.0) 2.1 Amplified
   BT-20 58 123 (2.1) 182 (3.1) 0.7 Not amplified
   MCF-7 59 123 (2.1) 165 (2.8) 0.8 Not amplified
Institut Curie, Paris, France
   SKOV-3 40 488 (12.2) 116 (2.9) 4.2 Amplified
   MDA-MB-453 40 388 (9.7) 128 (3.2) 3.0 Amplified
   BT-20 40 76 (1.9) 88 (2.2) 0.9 Not amplified
   MCF-7 40 108 (2.7) 112 (2.8) 1.0 Not amplified
CEP 17, centromere of chromosome 17 probe.

Study Center/Cell Line	No. of Nuclei Counted	Total Signals (Mean per Nucleus)	HER-2/CEP 17 Ratio	Conclusion
HER-2/neu	CEP
University of Wales Hospital, Cardiff
SKOV-3	59	571 (9.6)	212 (3.6)	2.7	Amplified
MDA-MB-453	60	504 (8.4)	237 (4.0)	2.1	Amplified
BT-20	58	123 (2.1)	182 (3.1)	0.7	Not amplified
MCF-7	59	123 (2.1)	165 (2.8)	0.8	Not amplified
Institut Curie, Paris, France
SKOV-3	40	488 (12.2)	116 (2.9)	4.2	Amplified
MDA-MB-453	40	388 (9.7)	128 (3.2)	3.0	Amplified
BT-20	40	76 (1.9)	88 (2.2)	0.9	Not amplified
MCF-7	40	108 (2.7)	112 (2.8)	1.0	Not amplified

Immunohistochemical Staining Characteristics of Cell Lines With Different Antibodies and Methods Used in Participating Breast Cancer Centers

Study Code Antibody* New Cell Line Control DAKO Cell Line Control
SKOV-3 MDA-MB-453 BT-20 MCF-7 SK-BR-3 (3+)^† MDA-175 (1+)^† MDA-231 (0)^†
A CB11 3 2 2 0 3 1 0
B CB11 3 2 0 0 3 0 0
C CB11 3 0 0 0 2 0 0
D 10A7 3 1 0 0 3 0 0
E HercepTest 3 2 1 0 3 1 0
F Polyclonal 3 2 1 0 3 1 0
G 3B5 3 2 0 0 3 0 0
* See Table 1.
† Expected level of staining for a valid result on the DAKO cell line control slide, as given in the HercepTest protocol (DAKO, Ely, Cambridgeshire, England).

Study Code	Antibody*	New Cell Line Control	DAKO Cell Line Control
SKOV-3	MDA-MB-453	BT-20	MCF-7	SK-BR-3 (3+)^†	MDA-175 (1+)^†	MDA-231 (0)^†
A	CB11	3	2	2	0	3	1	0
B	CB11	3	2	0	0	3	0	0
C	CB11	3	0	0	0	2	0	0
D	10A7	3	1	0	0	3	0	0
E	HercepTest	3	2	1	0	3	1	0
F	Polyclonal	3	2	1	0	3	1	0
G	3B5	3	2	0	0	3	0	0

Correlation of HER-2/neu Protein Overexpression by Immunohistochemical Analysis With HER-2/neu Gene Amplification by FISH

Cell Line HER-2/neu Status
Gene Amplification Protein Overexpression
3+ 2+ 1+ 0 Total
SKOV-3 Amplified 7 0 0 0 7
MDA-MB-453 Amplified 0 5 1 1 7
BT-20 Not amplified 0 1 2 4 7
MCF-7 Not amplified 0 0 0 7 7
FISH, fluorescence in situ hybridization.

*	DAKO, Ely, Cambridgeshire, England; Life Sciences International, Basingstoke, Hampshire, England; Menarini Diagnostics, Finchampstead, Berkshire, England; Novocastra Laboratories, Newcastle upon Tyne, England; Oncogene Research Products, CN Biosciences UK, Nottingham, England; Vector Laboratories, Peterborough, England.
†	Performed at room temperature unless stated otherwise.
‡	All procedures performed as directed in the HercepTest kit.

*	See Table 1.
†	Expected level of staining for a valid result on the DAKO cell line control slide, as given in the HercepTest protocol (DAKO, Ely, Cambridgeshire, England).

Cell Line	HER-2/neu Status
	Gene Amplification	Protein Overexpression
	Gene Amplification	3+	2+	1+	0	Total
SKOV-3	Amplified	7	0	0	0	7
MDA-MB-453	Amplified	0	5	1	1	7
BT-20	Not amplified	0	1	2	4	7
MCF-7	Not amplified	0	0	0	7	7