Prognostic Significance of Co-Expression of c-erbB-20ncoprotein and Epidermal Growth Factor Receptor in Breast Cancer Patients Akihiko Osaki, MD, Masakazu Toi, MI), Hirofumi Yamada, MD, Hiroyuki Kawami, MD, Katsumasa Kuroi, MD, Tetsuya Toge, MD, Hiroshima,Japan

The expression of c-erbB-2 oncoprotein and epidermal growth factor receptor (EGFR) was examined by immunocytochemical and radioreceptor assays in 1 1 5 patients with primary breast cancer. In 4 8 of 115 patients ( 4 2 % ) , the assays were found to be positive for the expression of c-erbB-2 oncoprotein, and, in 4 4 of 115 (35%) patients, the assays were positive for the expression of EGFR. There was no correlation between the expression of c-erbB-2 oncoprotein and EGFR. Clinical survey demonstrated that both c-erbB-2 oncoprotein expression and EGFR expression have independent prognostic values. Furthermore, when patients were divided into three groups on the basis of the expression of both c-erbB-2 oncoprotein and EGFR, those who were found to be positive for the expression of both c-erbB-2 oncoprotein and EGFR showed a worse prognosis than other groups. These results suggest that the combination of the expression of both c-erbB-2 oncoprotein and EGFR may be important in selecting patients who have a poor prognosis.

From the Department of Surgery, Research Institute for Nuclear Medicine and Biology, Hiroshima University, Hiroshima, Japan. Requests for reprints should be addressed to Akihiko Osaki, MD, Department of Surgery, Research Institute for Nuclear Medicine and Biology,Hiroshima University, Kasumi, 1-2-3, Minami-ku, Hiroshima, Japan 734. Manuscript submitted April 30, 1991, and accepted in revised form September 27, 1991.

he c-erbB-2 or neu oncogene encodes a glycoprotein T that is structurally similar to, but distinct from, the epidermal growth factor receptor (EGFR) [1] and is a member of the tyrosine kinase oncogene family. It is assumed to play a role in controlling cellular growth as do other growth factor receptors [2]. Amplification of the gene, which is found on human chromosome 17 [3], has been demonstrated in adenocarcinomas arising at a number of different sites [4,5]. In human breast cancer, the expression has been found in 10% to 33% of patients and has been related to short survival times and a poor prognosis [6-11]. EGFR status is also considered to be an independent prognostic indicator [12] and closely related to the hormone-independent growth of human breast cancer. Interaction between EGFR and c-erbB-2 has been examined in tumor cell lines [13,14]; however, little is known about its clinical significance. Therefore, we have compared the expression of c-erbB-2 oncoprotein and EGFR and examined the prognostic significance of each. MATERIALS AND METHODS Tissue specimens from 115 patients with primary breast cancer who underwent either standard radical mastectomy or modified radical mastectomy with full dissection of axillary lymph nodes were used for the study. Formalin-fixed paraffin-embedded tissues were used for the c-erbB-2 oncoprotein assay. Tissues frozen at -70~ were used for the EGFR assay. Both assays were performed with tissue from all patients. Immunocytochemical assay: The expression of c-erbB-2 oncoprotein was assessed by the avidin-biotin complex immunoperoxidase assay. Polyclonal antibody (Nichirei KK, Tokyo), raised in rabbits by immunization with a synthetic peptide corresponding to amino acid residues 1,242 to 1,255, which are found in the cytoplasmic domain of the predicted protein, was used [1,15], The immunocytochemical reactivity was graded according to the percentage of c-erbB-2 protein-stained cells found. Tissue with more than 50% stained tumor cells was designated as (+++), that with 25% to 50% (++), 1% to 25% (+), and negative (-). Tissues with a rating of (+) or more were defined as positive. Immunostaining of monoclonal antibody Ki-67, which detects the nuclear protein of cycling cells except those in the Go stage, was performed in 69 of 115 tumors. The number of Ki-67-positive tumor cells was determined in more than 2,000 tumor cells of the tissue section. Binding assay: The biochemical competitive binding assay for EGFR was performed as described previously

THE AMERICAN JOURNAL OF SURGERY VOLUME 164 OCTOBER 1992 39,3

O S A K I E T AL

TABLE

% 100

I

Secondary Factors

c-erbB-2(-) ( n : 67 )

c-erbB-2 0ncoprotein*

Items

Positive

Negative

p Value

28 (58)* 20 (42)

34 (51) 33 (49)

NS

30 (63) 18 (37)

33 (49) 34 (51)

NS

7 (15) 27 (56) 14 (29)

15 (22) 37 (55) 15 (22)

26 (54) 10 (15) 12 (21)

36 (54) 19 (28) 12 (18)

6 (13) 42 (87)

2 (3) 65 (97)

20 (42) 9 (19) 19 (39)

24 (36) 16 (24) 27 (40)

21 (44) 26 (54) 1 (2)

21 (31) 43 (64) 3 (5)

NS

27 (56) 21 (44) 48 (100)

43 (64) 24 (36) 67 (100)

NS

'g c-erbB-2(~)

Age (y) 50

Menopause Before After Tumor size (cm) 0-2 2-5 >5

Histology Noninvasive Invasive Immunoreactivity of Ki-67 (%)1 8

Unknown

0

i

i

1

NS

NS

)

2

i

~

i

3

4

5 year

Figure 1. The correlation of c-erbB-2 oncoprotein expression and postoperative relapse-free survival rate. A significant difference was found between patients who were positive for c-erbO-2 oncoprotein and those who were negative at 43 months after operation (p < 0 . 0 5 ) . Solid fine: c-erbB-2 oncoprotein positive; dotted fine: c-erbB-2 oncoprotein negative.

NS % 10(

E6FR(-) ! n = 71 )

m

EGFR

Negative Positive Total

= 48

Postoperatilb period

ER

Negative Positive Unknown

n

~p4

(

50

L

"g

1

EGFR(§ ( n = 44 ) 50

NS = not significant:ER = estrogen receptor; EGFR = epidermal

growthfactorreceptor. *Valuesin parenthesesare pereentages. VTheproportionof Ki-67-positivecells. ~p