J Cancer Res Clin Oncol (1992) 118:468-473

C iiicer mesearch Clinical 9 9 Springer-Verlag1992

Alterations of the c-erbB2 gene in human breast cancer B. Zoll 1, B. Kynast 2, B. Corell a, D. Marx 3, G. Fischer 3, and A. Schauer 3 1 Institut fiir Humangenetik der Universit~it Gfttingen, GoBlerstr. 12d, W-3400 G6ttingen, Federal Republic of Germany 2 Chirurgische Onkologie der Universit~t G6ttingen, Robert-Koch-Str. 40, W-3400 G6ttingen, Federal Republic of Germany 3 Pathologisches Institut der Universitfit G6ttingen, Robert-Koch-Str. 40, W-3400 G6ttingen, Federal Republic of Germany Received 12 August 1991/Accepted 11 December 1991

Summary. D N A amplification, R N A overexpression and p185 protein expression of the c-erbB2 oncogene were investigated in 109 cases of breast cancer with the aim of evaluating any correlation between the different methods. A correlation between Southern blotting and immunohistochemical analysis of paraffin-embedded material was found. Thus, amplification of the c-erbB2 oncogene leads to overexpression of the p l 8 5 protein. By contrast, no statistical correlation could be shown between R N A overexpression, measured by Northern blotting, and immunohistochemical p185 membrane stainings. It is o f special interest that most of the cases that are positive for Northern blotting and negative for immunochemistry are negative for Southern blotting as well. Contradictory findings between R N A overexpression and lack of immunohistochemical staining of p185 give rise to the assumption that a defective protein is encoded, which cannot be incorporated into the substructures of the tumour cell membrane. When screening for point mutations in the transmembrane domain of the c-erbB2 oncogene, no point mutation could be detected, either by using the endonuclease FokI, which cuts at position 2012 (the point mutation in the neu gene of the rat), or by direct sequencing.

Key words: c-erbB2 oncogene - c-erbB2 point mutation Breast cancer - Oncogenesis

Introduction In 1987 the first reports of a study of the correlation of relapse and survival with amplification of an oncogene in breast cancer were published (Slamon et al. 1987). Independently, different groups isolated a c-erbB-related gene called the H E R - 2 (Coussens et al. 1985) or erbB2 gene Abbreviations: PCR, polymerase chain reaction. Offprint requests"to: B. Zoll

(Schechter et al. 1985) by screening a human c D N A library with c-erbB probes. This gene is homologous to the rat neu gene (Yamamoto et al. 1986) and is localized on chromosome 17q21-22 (Fukushige et al. 1986). It contains seven exons and codes a transmembrane glycosylized phosphoprotein of 185 k D a with tyrosinase activity (Akiyama et al. 1986). Different mechanisms for activating the gene are known, and the normal rat gene and the transforming counterpart differ only by a point mutation (Bargrnann et al. 1986) at position 2012, where a change from T to A is observed. An amplification of the c-erbB2 gene was ascertained in different human tumour cell lines (Fukushige et al. 1986). The intention of our study was to compare the alterations of the c-erbB2 gene, the amplification, the R N A overexpression, the p185 expression and the existence of a point mutation with regard to the oestrogen receptor status and the histology of the tumour tissues.

Materials and methods We tested 100 samples of breast neoplasms for gene amplification, 56 samples for RNA overexpression and 93 sections of tissue, which had been fixed in formalin and embedded in paraffin, for p185 overexpression. Polymerase chain reaction (PCR) products of the transmembrane region of the c-erbB2 gene were produced from 100 breast tumours and screened for point mutations at position 2012; 20 PCR products were directly sequenced. The diagnoses, oestrogen receptor status and histological analyses were classified by pathological investigations in the clinics. After surgery, tissue samples were immediately frozen in liquid nitrogen. The diagnoses were made by clinicians, the classification of oestrogen receptor status and the histological analyses by pathological investigations. DNA amplification. DNA extraction was carried out using techniques described previously (Corell and Zoll 1988a). DNA (7.5 ~tg) was digested with EcoRI, separated on 0.8% agarose gels and transferred to nitrocellulose filters by the Southern procedure (Southern 1975). Blots were hybridized to the 32p-labelled pliER2-436 plasmid, carrying a 1.6 x 103-base(l.6-kb) EcoRI fragment, which represented the first five exons of the c-erbB2 gene (Coussens et al. 1985). To control the amount of DNA applied to each lane a rehybridization or double hybridization with a 32p-labelled 2.6-kb

469 EcoRI fragment recognizing the 3' end of the c-mos locus (Corell and Zott 1988 b) was done. The intensity of the resulting signals was determined by densitometry. RNA overexpression. Tumour biopsies were shredded in liquid nitrogen. RNA extraction was done as described by Chirgwin et al. (1979). Electrophoresis was on formaldehyde gels and RNA was checked by staining with ethidium bromide. After RNA transfer onto nitrocellulose filters, blots were hybridized to a 32p-labelled pCER-204 plasmid (Yamamoto et al. 1986; Maniatis et al. 1982). To determine the amount of RNA in each lane a rehybridization with a n a-actin probe (Hanauer et al. 1983) was carried out. On each filter, RNA from a mammary tumour cell line BT20 with normal cerbB2 expression (Kraus et al. 1987) was run.

purified using Geneclean, and 20 PCR products from the breast cancer DNA were directly sequenced using the appropriate primers by the method of Sanger (Sanger et al. 1977).

Results F r o m a total of 100 tested D N A samples of breast neoplasms, 19 showed an amplification (19%) (Fig. 1). No amplification was found in the D N A o f white blood cells from the same patients - this can be assessed as a tumour-specific phenomenon. The correlation between

Immunohistochemical analysis. For the purpose of immunohistochemical analysis we used the c-erbB2-specific monoclonal antibody acting against the external domain (9G6, Oncogene Science Dianova, Hamburg) and against the internal domain (3B5, Oncogene Science Dianova, Hamburg; Van de Vijver et al. 1987). Screening for point mutations. A 272-bp fragment of the c-erbB2 gene containing the coding region of the transmembrane domain was amplified by means of the PCR method (polymerase chain reaction) (Saiki 1989) and, by means of the specific cutting enzyme FokI, screened for the point mutation known in the neu oncogene. PCR amplification used the following oligonucleotides, which flank the transmembrane domain, as primers: BC9, 5' CAT GCC AGC CTT GCC CCA TCA ACT; BC10, 5' CAT CTG CGC CTG GTT G G G CAT CGC. The oligonucleofides were synthesized in the Max Planck Institute of Biophysical Chemistry, G6ttingen. The reaction mixture consisted of 0.5 ~tg genomic DNA, 5 Ixl polymerase buffer (10-fold) 5 ~tl dimethylsulphoxide, 1.25 mmol each dNTP and 50 pmol each primer, made up to 50 ~tl with double-distilled water. Before undergoing 35 cycles of PCR denaturation at 95 ~ C for 45 s, annealing at 55 ~ C for 45 s and elongation at 63 ~ C for 3 rain in a Biomed Thermocycler, the reaction mixture was incubated at 95 ~ C for 7 rain. A 3-~tl sample of the amplification product was electrophoresed on a polyacrylamide gel and visualized under ultraviolet light after ethidium bromide staining. PCR-amplified material was

Fig. 2. Overexpression of c-erbB2 in mammary carcinomas. RNA of six different tumours (T1-T6) and of the mammary carcinoma cell line BT 20 (MC) (Kraus et al. 1987) was isolated and hybridized with pCER 204. The RNA of tumour T4 shows a 2- to 5-fold increased c-erbB2 level. The expression of tumour T5 was estimated separately as being more than 10-fold

Table 1. Copies of the c-erbB2 gene in relation to the oestrogen

receptor (ER) status and clinical status in breast cancer" Parameter

Fig. 1. Gene amplification for c-erbB2 (lane 6) in a mammary carcinoma. DNA of white blood ceils (B) and tumour tissue (73 of four different patients was digested with EcoRI and double-hybridized with plier2-436 (Coussens et al. 1985) and c-mos (Corell and Zoll 1988 b). The 6.6-kb and 4.6-kb fragments correspond to the c-erbB2 and the 2.6-kb fragment to the c-mos signal

All turnouts investigated

No amplification

Amplification

n

n

n

Receptor status ER + + ER + ER (+) ER-

91 b

Clinical status I 11 III

88 b

17 28 23 23

27 55 6

(%)

(%)

74 (18.7) (30.7) (25.3) (25.3)

16 22 18 18

17 (21.6) (29.8) (24.3) (24.3)

74 (30.7) (62.5) (6.8)

23 46 5

(%)

1 6 5 5

(6.9) (35.3) (29.4) (29.4)

14 (31.1) (62.2) (6.7)

4 9 1

(28.6) (64.3) (7.t)

a Receptor status: ER + +, elevated level > 100 fmol/ng protein; ER +, positive 20-100 fmol/ng protein; ER (+), decreased level 5-19 fmol/ng protein; E R - , negative < 5 fmol/ng protein. Clinical status: classification after the TNM system b Clinical data were not available from all tumours investigated

470 Table 2. Expression of the c-erbB2 gene in relation to prognostic parameters Parameter

Normal expression

Expression 2- to 5-fold

ExpresExpression sion 5- to 10-fold > 10-fold

n

n

n

Receptor status E R ++ ER + ERt+) ER-

41 b

Clinical status I II III

40 b

11 12 10 8

12 25 3

(%)

(%)

11 (26.8) (29.3) (24.4) (19.5)

4 5 1 1

(36.4) (45.4) (9.1) (9.1)

10 (30.0) (62.5) (7.5)

(%)

n

2

1

1 1

1

(50) (50)

1

3 6 1

(30.0) (60.0) (10.0)

1

(%)

(100)

1 I

(100)

(100)

Receptor status: E R ++, elevated level > 100 fmol/ng protein; E R +, positive 20-100 fmol/ng protein; E R (§ decreased level 5-19 fmol/ng protein; E R - , negative < 5 fmol/ng protein. Clinical status: classification after the T N M system b Clinical data were not available from all tumours investigated

Table 3. Correlation between D N A amplification and R N A overexpression (Sn = 49) Parameter

DNA amplification

No D N A amplification

n

(%)

n

(%)

R N A overexpression No R N A overexpression

5 -

(10.2) -

6 38

(12.2) (77.6)

Total

5

44

DNA amplification and the oestrogen receptor status and the clinical status did not show any significant difference (Xz test) with respect to the low number of cases (Table 1). R N A was extracted from 56 tumours. In 15 samples (27%) RNA overexpression was ascertained and, by means of a dilution series, was subdivided into 2to 5-fold, 5- to 10-fold and > 10-fold expression (Fig. 2). A comparison of the c-erbB2 expression with the oestrogen receptor status and the clinical status, in view of the pathological staging, did not show any statistically significant differences (Table 2). Out of the total number of tested tumours, 49 could be compared with regard to their DNA amplification and R N A overexpression. Correlation between D N A amplification and R N A overexpression was found in 5 of 49 tumours (10.2%), whereas an R N A overexpression without D N A amplification was found in no fewer than 6 tumours (12.2%). For 5 patients both D N A amplification and R N A overexpression were found; for 6 cases R N A overexpression without DNA amplification was demonstrated. We could not find any case with DNA amplification that lacked R N A overexpression (Table 3). An immunohistochemical analysis was carried out on the sections of 93 breast neoplams: 76 (81.7%) turned out negative; 17 (18.3%) were found positive (Figs. 3, 4). D N A amplification, R N A overexpression and p185 overexpression could be examined on 42 tumours (Table 4): 32 tumours (76.2%) turned out negative in respect of all parameters examined; 1 tumour (2.4%) was positive in all aspects; 6 of the tumours (14.3%) had no D N A amplification but R N A overexpression and no p185 overexpression. An association could be shown between D N A amplification and p185 overexpression. Out of 14 cases 11 were positive not only for Southern blotting but also for immunohistochemistry (Table 5). On the other hand there

Fig. 3. Invasive ductal carcinoma with p185 overexpression (membrane staining); incubation with a monclonal antibody directed against the external domain of the tumour cell membrane

471

Fig. 4. Invasive ductal carcinoma with nonspecific cytoplasma staining; incubation with a monoclonal antibody directed against the internal domain of the tumour cell membrane Table 4. Correlation between DNA amplification, RNA overexpression and p185 overexpression (Sn = 42)

Table 6. Correlation between RNA overexpression and pl85 overexpression (Sn=45)

Parameter

Parameter

RNA overexpression p185 overexpression No RNA overexpression p185 overexpression RNA overexpression no p185 overexpression No RNA overexpression no p185 overexpression Total

DNA amplification

No DNA amplification

n

(%)

n

(%)

1

(2.4)

-

-

1

(2.4)

1

(2.4)

1

(2.4)

6

(14.3)

-

-

32

(76.2)

RNA overexpression

No RNA overexpression

n

(%)

n

(%)

p185 overexpression No p185 overexpression

2 6

(4.4) (13.3)

2 35

(4.4) (77.9)

Total

8

Table 7. Correlation between the histology of the tumours and

c-erbB2 alterations a 3

39

Carcinomas

n

p185 overexpression No p185 overexpression Total

DNA amplification

No DNA amplification

n

(%)

n

(%)

11 3

(12.4) (3.4)

3 72

(3.4) (80.8)

14

75

was no positive correlation between RNA overexpression a n d p 1 8 5 o v e r e x p r e s s i o n ( T a b l e 6). C o m p a r i n g t h e r e s u l t s o f t h e h i s t o l o g i c a l a n a l y s e s to DNA amplification, RNA overexpression and p185 overexpression, no statistically significant correlation w a s f o u n d ( T a b l e 7). Since m o s t o f t h e t u m o u r s i n v e s t i g a t e d were i n v a s i v e d u c t a l c a r c i n o m a s , we o b s e r v e d m o l e c u l a r a l t e r a t i o n s m o s t f r e q u e n t l y i n these t u m o u r s .

c-erbB2

Additional in situ carcinomas

TableS. Correlation between DNA amplification and p185 overexpression (2;n = 89) Parameter

37

DNA amplification

RNA overexpression

p185 overexpression

13 -

14 1 -

-

15 1 1 . -

1 1

1 1

1

17

15

17

DCIS

CLIS 1 -

ID LOB SC MUC SR TUB MED

83 9 5 3 2 3 4

6 . -

Total

109

6

.

.

.

a ID, invasive ductal carcinoma; LOB, lobular carcinoma; SC, scirrhous carcinoma; MUC, mucinous carcinoma; SR, signet-ring cell carcinoma; TUB, tubular carcinoma; MED, medullary carcinoma. DCIS, ductal carcinoma in situ; CLIS, lobular carcinoma in situ S c r e e n i n g o f 100 P C R p r o d u c t s o f t h e t r a n s m e m b r a n e r e g i o n o f t h e c-erbB2 g e n e for t h e FokI c u t t i n g site, w h i c h a p p e a r s i n c o n n e c t i o n w i t h a p o i n t m u t a t i o n , i n n o case showed any deviation from the normal fragment length.

472

Fig. 5. Direct sequencing of polymerase chain reaction products of the 272-bp c-erbB2 region. The part that contains the point mutation in the rat neu oncogene is marked with brackets

The subsequent direct sequencing of the 272-bp fragment of the transmembrane region has not shown a point mutation (Fig. 5).

Discussion The value of the c-erbB2 investigations regarding oncogenesis and prognosis is a subject of controversy. Some investigators confirm a connection between the c-erbB2 amplification and the prognosis (Zeilinger et al. 1989). In our study, gene amplification was found in both the advanced and early stages of disease, therefore the c-erbB2 gene may possibly be regarded as an important factor of oncogenesis in a subgroup of human breast cancer. Since our patients were investigated during the last 2 years we cannot yet suggest a prognosis, Some studies have shown that an amplification does not necessarily correlate with an overexpression (Van de Vijver et al. 1987). In the latest reports (Borg et al. 1991) an association between c-erbB2 amplification, a high proliferation rate and a poor prognosis could be demonstrated in steroid-receptor-positive tumours but not in receptor-negative tumours, whereas members of our study group (Marx et al. 1990) have shown a positive correlation of p185 expression - measured by monoclonal antibodies and histological grade-but a negative correlation with steroid receptor status. It seems that gene alterations do not depend on the histology of the turnouts. No correlation to the histological subtypes was observed but here the low number of tumours investigated has to be taken into account. In our

study we ascertained an overexpression without prior amplification in 12.2% of the tumours tested. Principally different mechanisms are conceivable as a reason for overexpression. On the one hand, translocations might result in changes of expression (Hooberman et al. 1989), on the other hand, expression-regulating sequences might have changed within the c-erbB2 gene. In some genes, e.g. the Ha-ras, c-mos and c-myc genes of the mouse (Blair et al. 1986), negative transcription elements have been found in the 5' region. After deletion of these regions the expression increases. Furthermore, it is known that the expression depends on the interaction of specific factors (e.g. trans-acting proteins) with promotor or enhancer elements (e.g. cis-acting regions) (Ostertag et al. 1987; Schtler et al. 1989). A change within these cis-acting regions or in the concentration of the trans-acting factors may also result in a deviation from the normal expression. Since the 5' region of the c-erbB2 gene has not been characterized yet, it is not known whether transcription-regulating regions exist. The difference in the concentration of R N A and p185 can be explained by the dilution effects in analyses of tumour tissues permeated with normal tissue. Using this method the measured R N A concentration is lower than that of protein measured directly on stained sections. A non-detectable p185 overexpression with R N A overexpression can be interpreted as a defective protein not recognized by the monoclonal antibody. A defective protein could arise because of a mutation in the coding region of the gene. Contrary to Borg et al. (1991), who reported a correlation to a poor prognosis, another group (De Potter et al. 1990) has found neu protein overexpression to be a predicting factor in the ability to create haematogenous metastases in human breast cancer patients. As our investigations give a correlation between Southern hybridization and immunohistochemistry this might be a suitable method on which to base a prognosis. A point mutation in the transmembrane region of the protooncogene, which is found to be a mechanism for transformation in the neu gene of the rat, seems not to play an important role in transformation of the human cerbB2 gene. Nevertheless, since we have screened only 20 tumours by directly sequencing the transmembrane region it is not impossible that a point mutation somewhere in the gene sequence may be responsible for malignant transformation. Further studies on, for example, the importance of p53, the so-called tumour-suppressor gene, are necessary to clarify tumorigenesis. As our investigations show a correlation between Southern hybridization and immunohistochemistry, this might be a suitable basis for a prognosis. Acknowledgements. Thanks are due to R. Bertram for technical help and I. Reise and M. DeseniBfor secretarial assistance. We thank A. Ullrich and T. Yamamoto for making the different Her-2 probes available to us. Further, the authors are grateful to A. Hanauer for the a-actin probe. We are indebted to the hormone laboratory of the Pathology Centrum of the Universit~itsklinikG6ttingen for examination of the oestrogen receptor level. We especiallythank T. Bauknecht, P. B6hme, E. Gallasch, M. Kneba, W. Rath and R. Rauskolb, who sent us blood samples and tumour tissues.

473

References Akiyama T, Sudo CH, Ogawara H, Toyoshima K, Yamamoto T (1986) The product of the human c-erbB2 gene: a 185 kilodalton glycoprotein with tyrosine kinase activity. Science 232:16441646 Bargmann CI, Hung MC, Weinberg RA (1986) Multiple independent activations of the oncogene by a point mutation altering the transmembrane domain of p185. Cell 45:649-657 Blair DG, Oskarsson MK, Seth A, Dunn KJ, Dean M, Zweig M, Tainsky MA, Vande Woude DF (1986) Analysis of the transforming potential of the human homolog of mos. Cell 6:785794 Borg A, Baldetorp B, Fern6 M, Killander D, Olsson H, Sigurdsson H (1991) ERBB2 amplification in breast cancer with a high rate of proliferation. Oncogene 6:137-143 Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ (1979) Isolation of biologically active fibonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294-5299 Corell B, Zoll B (1988 a) Comparison between the allelic frequency distribution of the Ha-ras 1 locus in normal individuals and patients with lymphoma, breast, and ovarian cancer. Hum Genet 79:255-259 Corell B, Zoll B (1988 b) Evidence against a tumour-specific EcoRI RFLP of the c-mos locus. FEBS Lett 230:81-84 Coussens L, Yang-Feng TL, Liao YC, Chen E, Gray A, McGrath J, Seeburg PH, Libermann TA, Schlessinger J, Francke U, Levison A, Ullrich A (1985) Tyrosine kinase receptor with extensive homology to EGF receptor shares chromosomal location with neu oncogene. Science 230:1132-1139 De Potter CR, Beghin C, Makar AP, Vandekerckhove D, Roels HUJ (1990) The neu-oncogene protein as a predictive factor for haematogenous metastases in breast cancer patients. Int J Cancer 45:55-58 Fukushige SI, Matsubara KI, Yoshida M, Sasaki M, Suzuki T, Semba K, Toyoshima K, Yamamoto T (1986) Localisation of a novel v-erbB-related gene c-erbB2 on human chromosome 17 and its amplification in a gastric cancer cell line Mol Cell Biol 6:955-958 Hanauer A, Levin M, Heilig R, Daegelen D, Kahn A, Mandel JL (1983) Isolation and characterization of cDNA clones for human skeletal muscle c~-actin. Nucleic Acid Res 11:3503-3516 Hooberman A, Carrino J J, Leibowitz D, Rowley Jo, Le Beau MM, Arlin ZA, Westbrook CA (1989) Unexpected heterogeneity of BCR-ABL fusion mRNA detected by polymerase chain reaction in Philadelphia chromosome positive acute lymphoplastic leukemia. Proc Natl Acad Sci USA 86:4259-4263 Kraus MH, Popescu NC, Ambaugh SC, King CR (1987) Overexpression of the EGF receptor-related proto-oncogene erbB-2 in human mammary tumor cell lines by different molecular mechanisms. Embo J 6:605-610

Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning. Cold Spring Harbor Press, Cold Spring Harbor Marx D, Schauer A, Reiche C, May A, Ummenhofer L, Reles A, Rauschecker H, Sauer R, Schumacher M (1990) C-erbB2 expression in correlation to other biological parameters of breast cancer. J Cancer Res Clin O ncol 116:15-20 Ostertag W, Stocking C, Johnson GR, Kluge N, Kollek R, Franz T, Hess N (1987) Transforming genes and target cells of routine spleen focus-forming viruses. Adv Cancer Res 48:193-355 Saiki RK (1989) The design and optimization of the PCR. In: Ehrlich HA (ed) PCR technology. M Stockton press, New York, pp 7-/6 Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 80:46794683 Schechter AL, Hung MC, Vaidyanathan L, Weinberg RA, YangFeng TL, Francke U, Ullrich A, Coussens L (1985) The neu gene: an erbb-homologous gene distinct from and unlinked to the gene encoding the EGF receptor. Science 230:976-978 Sch61er HR, Hatzopoulos AK, Balling R, Suzuki N, Gruss P (1989) A family of octamer-specific proteins present during mouse embryogenesis: evidence for germline-specific expression of an Oct factor. EMBO J 8:2543-2550 Slamon D J, Clark GM, Wong SG, Levin WL, Ullrich A, McGuire WL (1987) Correlation of relapse and survival with amplification of the her-2/neu oncogene. Science 235:177-182 Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel eleetrophoresis. J Mol Biol 98:503517 Vijver MJ van de, Bersselaar R van de, Devilee P, Cornelisse C, Peterse JL, Nusse R (1987) Amplification of the neu (c-erbB-2) oncogene in human mammary tumors is relatively frequent and often accompanied by amplification of the linked c-erbA oncogene. Mol Cell Biol 7:2019-2023 Vijver MJ van de, Peterse JL, Mooi WJ, Wisman P, Lomans J, Dalesio O, Nusse R (1988) Neu-protein overexpression in breast cancer: association with comedo-type ductal carcinoma in situ and limited prognostic value in stage II breast cancer. N Engl J Med 19:1239-1245 Yamamoto T, Ikawa S, Akiyama T, Semba K, Nomura N, Miyajima N, Saito T, Toyoshima K (1986) Similarity of protein encoded by the human c-erbB-2 gene to epidermal groth factor receptor. Nature 319:230-234 Zeilinger R, Kury F, Czerwenka EK, Sliutz G, Knogler W, Huber J, Zielinsky C, Reiner G, Jakesz R, Staffen A, Reiner A, Wrba F, Spona J (1989) Her-2 amplification, steroid receptors and epidermal growth factor receptor in primary breast cancer. Oncogene 4:109-114