Cytogenetics of breast cancer

Cytogenetics of Breast Cancer Detlef Geleick, Hansjakob Mtiller, Alex Matter, Joachim Torhorst, and Urs Regenass

ABSTRACT: Chromosome counts were pedormed on 1,100 cells ]rom 17 malignant breast carcinomas and on 168 cells of four normal tissue samples a~ter amethopterin treatment and G-banding. Karyotypes were established from 216 cells of 11 tumor-derived cultures and from 47 cells of four nonmalignant tissue-derived cultures. Karyotypes of cells from nonmalignant samples showed a normal diploid chromosomal constitution with no consistent loss or gain of a specific chromosome. Structural chromosomal abnormalities were not observed. Tumor-derived cultures could be distinguished from normal cultures on the basis of a significantly increased incidence of numerical changes and structural chromosomal aberrations. In nine of 11 tumorderived cultures, numerically normal cells were shown to be pseudodiploid, with frequencies ranging to 43% (mean, 13.2%) of the diploid cells. In agreement with previous reports, cytogenetic analyses showed predominantly diplaid cells. Chmul numerical changes of chromosomes !7, 18, 20. and 21 could be detected in three tumor samples. Chmal structural abnormalities could be observed in two of 11 analyzed tumours. A t(6;12)(p21;p13J and an enlarged chromosome 7 (7q ÷ I were found in a patient with invasive ductal carcinoma. An inversion af chromosome 7 [inv(7)(ql 1.2q32) 1 was observed in one case. also diagnosed us invasive ductal carcinoma. The significance of these findings in relation to clinical data is discussed.

INTRODUCTION C o n s i s t e n t c y t o g n e t i c m a r k e r s h a v e b e e n i d e n t i f i e d in a v a r i e t y of t u m o r s ]1 3]. A l t h o u g h h u m a n b r e a s t c a n c e r c a n n o t yet be a s s o c i a t e d w i t h u n i q u e k a r y o t y p i c c h a n g e s , a n u m b e r of r e p o r t s h a v e d e s c r i b e d t h e n o n r a n d o m i n v o l v e m e n t of c e r t a i n c h r o m o s o m e s . C h r o m o s o m e 1 ( l q ) w a s s h o w n to be f r e q u e n t l y i n v o l v e d in b o t h s t r u c t u r a l a n d n u m e r i c a l c h r o m o s o m a l a l t e r a t i o n s [ 4 - 1 0 ] . C h r o m o s o m e s 6, 7, a n d 11 w e r e s h o w n to b e a l t e r e d i n m o s t b r e a s t t u m o r s [8, 10]. Losses of c h r o m o s o m e s 8, 13, a n d 16 in n e a r d i p l o i d t u m o r s [7, 10] a n d losses of c h r o m o s o m e s 8 a n d 13 in p o l y p l o i d t u m o r s w e r e n o t e d [11]. A 3p d e l e t i o n a n d a c l o n a l t(1;4) in p r i m a r y b r e a s t c a r c i n o m a a n d a c l o n a l t(1;5) in a m e t a s t a t i c t u m o r w e r e d e s c r i b e d r e c e n t l y [12]. R e s t r i c t i o n f r a g m e n t l e n g t h p o l y m o r p h i s m (RFLP) s t u d i e s h a v e d e t e c t e d n o n r a n d o m d e l e t i o n s of c h r o m o s o m e s 11 [13], 13, a n d o t h e r s [14]. A loss of h e t e r o z y g o s i t y of c h r o m o s o m e 17 w a s d e m o n s t r a t e d r e c e n t l y [15]. C y t o g e n e t i c o b s e r v a t i o n s so far h a v e s h o w n a n u n o b t r u s i v e p i c t u r e of c h r o m o s o m e 17. C h r o m o s o m a l e v i d e n c e of g e n e a m p l i f i c a t i o n

From the Research Department, Pharmaceuticals Division, Ciba-Geigy (D. G.. A. M., U. R.), the Laborat(~ry of Human Genetics, the Department of Research of the University Clinics, Kantonsspitat Basel, (Lt. M.). and the Department of Pathology, University of Basel ( J. T.), Basel, Switzerland. Address reprint requests to: Urs Regenass, (Ph.D., Pharmaceuticals Division, Ciba-Geigy Ltd., Basel CH-4002, Switzerland. Received December 21, 1987; accepted October 18, 1989.

217 (el 1990 Elsevier Science Publishing Co., Inc. 655 Aw~nue of the Americas, New York, NY 10010

Cancer Genet Cytogenet 46:217-229 (1990) 0165-4608/90/$03.50

218

D. Geleick et al.

Relative Frequency % 8O i

!

Normal Tissue Tumor Tissue MM

°° i i

40

20

0 36

38

40

42

,t.4

46

48

50

52

Number of Chromosomes Figure 1 Number of cells with a given amount of chromosomes relative to the total number of cells analyzed. Total number of tumor tissue-derived cells 1,100; total number of normal tissue-derived cells 168. such as double m i n u t e s (dmin) and h o m o g e n e o u s l y staining regions (HSR) has been reported for several h u m a n breast tumors [10, 16-19]. The cytogenetic data base for h u m a n breast cancer is still limited owing to difficulties in obtaining high numbers of good-quality karyotypes. Recently described and improved culture conditions for primary breast cancer cells [20] allowed analysis of a large n u m b e r of karyotypes. These new techniques have been a p p l i e d in only a few recent studies [12, 21-23], resulting in p r e d o m i n a n t l y d i p l o i d cells with no or only rare clonal aberrations. We have extended the studies on the chromosomal distributions and karyotypes of p r i m a r y breast cancer in short-term cultures. In contrast to previous studies [12, 23], tissue cultures were initiated from the total tumor digest to m i n i m i z e tumor cell loss by organoid separation and to increase the chance of growth of a representative tumor cell population. Cytogenetic data obtained for tumor-derived cells were compared with those derived from cells of healthy breast tissue. Clonal karyotypic aberrations not observed in previous studies have been identified.

MATERIALS AND METHODS Tissue Collection We analyzed 17 h u m a n breast tumor samples (MaCa) and four n o n t u m o r o u s tissue samples (Mp). In two cases (MaCa 33, MaCa 41), the patients had a first-degree relative diagnosed with breast cancer. None of the patients had any therapy before surgery. The patients' data are s u m m a r i z e d in Table 1.

1

58 57

62

45 68 55 71 64 65

69 44 70

M a C a 40 M a C a 41

M a C a 47

50 55 57 83 85 86

MaCa MaCa MaCa MaCa MaCa MaCa

M a C a 87 M a C a 88 M a C a 89

Post Pre Post

Pre Post Post Post Post Post

Post

Post Hysterectomy

Post Pre Pre Post Hysterectomy

Menopausal status

data

4 4 3

8 3 3.5 NA NA 3

5.5

1.7 2.6

4.3 2.3 1.5 NA 1.5

Tumor size (in cm)

IDC IDC IDC

[LC IDC IDC IDC IDC IDC

IDC

MC IDC

SDC IDC IDC IDC IDC

Histology

NA NA III

111 llI II| NA NA 111

lII

II1 liI

III Ill NA NA II

Grading - / + /+ + / + + / +

+ + -

/ + /

+ /+

+/+ +/+ +/+ + / NA + /

+ !+ /+ +/+

+ +

+

+ + + + +

+ + +

+ /+

ER/PR

Node involvement

NA ND

NA

13.9 ND ND NA NA ND

ND

13 3.2

ND ND NA NA 3

EGF-R

NA NA

NA

0 0 0 NA NA NA

0

0 Sister

0 Mother NA NA 0

Relatives with breast cancer

NA A l i v e at 27 m o

progressive, a l i v e at 22 m o Local recurrence 12 too, c a n c e r progressive, a l i v e at 27 m o A l i v e at 29 m o A l i v e at 27 m o A l i v e at 32 m o NA NA Bone marrow metastasis: alive a t 22 m o NA

1"1 n l o ; c a n c e r

A l i v e at 34 m o A l i v e at 42 m o NA NA Cancer contralateral, alive at 43 m o A l i v e at 3 m o Local recurrence

Subsequent course

Abbreviations: ER. estrogen receptor; PG, progesterone receptor: EGF-R, epidermal growUl factor receptor (fmol/mg protein): SDC. solid, partial dissolute carcinoma: ND, not done; IDC, invasive ductal carcinoma; NA, data not available; MC. medullary carcinoma; ILC. invasive lobulary carcinoma.

56 50 46 72 43

Age (yr)

Patient

30 33 34 37 39

MaCa MaCa MaCa MaCa MaCa

Sample (code)

Table

¢,D

b,3

220 Table 2

t~. Geleick et al.

Chromosome counts in metaphase spreads

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23

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32

33

34

35

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44

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17 17 17

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12 21

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21

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25 25

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21 1 I)

MilCa 3!t

19

M a C a 4(1

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MaCa 47

12 25

MaCa 50

21

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16

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14 43 16

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15 15

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15

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Except for MaCa 30, which probably represented a local recurrence of an invasive duct breast cancer, all malignant tissues were obtained from primary breast cancers. Histopathologically normal tissues were obtained from areas distal to the primary tumors or from reduction mammoplasties. Cells and Culture Techniques Tissue samples were processed as described by Stampfer et al. [24]. To avoid loss of single cells, the tumor tissue fragments were not collected by filtration on polyesterscreen filters. Cells were grown in plastic culture flasks in m e d i u m MM [25] designed for human mammary epithelial cells. When numerous mitotic figures were present in the cultures, they were either treated directly with alnethopterin and Colcemid or dissociated with trypsin-EDTA and subcultured before treatment. The cell lines for the conditioned media were obtained from the American Type Culture Collection (Rockville, MD). All cultures, except MaCa 30, exhibited a typical epithelial morphology of the cuboidal type, A recent i m m u n o h i s t o c h e m i c a l analysis using anticytokeratin antibodies verified the epithelial nature of these cells ]26]. Cells derived from MaCa 30 were spindle-shaped in morphology and negative for cytokeratins [20]. The nature of these cells could not be determined unequivocally. C h r o m o s o m e Harvest and Analysis Subconfluent cultures were amethopterin treated and processed after a method of Yunis and Chandler [27]. Slides were made by the conventional air-drying method. Chromosome counts were performed on Giemsa-banded metaphases. The metaphases for karyotyping were randomly selected in the range of 44 49

2 21

Cytogenetics of Breast Cancer

Metaphase plates with

48

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161

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65.8

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2118

61

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82

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c h r o m o s o m e s where the most prominent differences in c h r o m o s o m e numbers between normal and tu/nor tissues were visible (Fig. 1). A m i n i m u m of 10 metaphases per case was karyotyped whenever possible (exception MaCa 89). Karyotypes were described according to the International System for Human Cytogenetic Nomenclature [28]. Receptor Determinations Estrogen (ER) and progesterone (PR) receptor content were determined by the dextrancharcoal method [29]. Tumor samples containing more than 10 fmol/mg protein were considered ER + and PR +, respectively. Epidermal growth factor receptors (EGF-R) were determined as described by Fabbro et al. [30]. Statistical Analysis Chromosome counts of normal and tumor-derived cultures were analyzed by Student's t test. RESULTS C h r o m o s o m e Counts in Metaphase Plates Chromosome counts were performed from 17 tumor-derived (MaCa) and four normal tissue samples (Mp). Data of the c h r o m o s o m e counts are shown in Table 2. The mean percentage of cells with 46 c h r o m o s o m e s was 72.7% (range 61.3 82.6%) in normal tissue-derived cultures as compared with 32% (range 5.5-75%) in tumorderived cultures. Both distribution curves were asymmetric toward lower chromo-

222

D. Geleick et al.

Table 3

Chromosome analyses of cultures derived from normal breast epithelial cells

Case

Days in culture

Total

Normal

Pseudodiploid

Aneuploid

Mp Mp Mp Mp

17 17 17 12

13 11 10 12

10 9 10 10

0 0 0 0

3 2 0 2

1 2 5 12

No. and karyotypes with identified structural abnormalities including clonal numerical changes 0 0 0 0

some numbers (Fig. 1). The n u m b e r of metaphases with less than 46 ch r o m o so m es and more than 46 c h r o m o s o m e s differed significantly between cells derived from tumor and healthy tissues (p = 0.05, p - 0.01 respectively). In general, tumor-derived cultures s h o w e d a striking h y p e r d i p l o i d y (chromosome n u m b e r more than 46 per metaphase excluding tetraploidy), whereas h y p e r d i p l o i d y was rarely observed in normal tissue-derived breast cultures (Table 2). Tetraploid cells could be detected in both types of culture to a similar extent. Only one tumor-derived culture (MaCa 89) showed a high percentage of cells with 46 c h r o m o s o m e s (75%) and a distribution pattern similar to that of normal tissuederived cultures (Table 2). With one exception (MaCa 37), cultured sp eci m en s showed Figure 2 Involvement of given chromosomes in loss, gain, and structural changes. Percentages represent number of patients with genomic aberrations in relation to the total number of patients (11) analyzed. In cases in which karyotypes were obtained from two different cultures (MaCa 33, MaCa 39), the patient was rated as positive when the aberration was found in at least one culture.

Relative Frequency % 70 L~M Structural change 60

Gain of chromosome Loss of chromosome

50 40 30 20 10 0

I

I

1 234

I

I

I

I

5

6

H I

I

m

9 10 1112 1 3 1 4 1 5 1 6 Chromosome Number

I

I

I

171819202122XX

I

223

Cytogenetics of Breast Cancer

Number of Cells 20 ~'M

Structural change Gain of chromosome Loss of chromosome

15

10

0

I

1 2

3

I

I

I

4 5 6 7 8

I

I

l

I

I

1314151617 9101112 Chromosome Number

I

18 19 20 21 22 XX

Figure 3 Involvement of given chromosomes in loss, gain, and structural changes. Karyotypes of 216 cells from 11 patients were analyzed for structural and numerical abnormalities.

a u n i m o d a l chromosomal distribution pattern with a definite mode at 45 or 46 chromosomes.

Numerical and Structural Aberrations in Karyotypes Eleven tumors and four normal breast tissue specimens were cytogenetically characterized. From MaCa 33 and MaCa 39 karyotypes from different culture periods were established. We did not observe a preferential loss or gain of any given chromosome in normal tissue-derived cultures. The modal chromosome n u m b e r was 46. No clonal chromosomal abnormality could be detected. All cells with 46 chromosomes were truly diploid (Table 3). Neither did tumor-derived cultures show a consistent pattern of loss and gain, but certain structurally normal chromosomes showed a tendency to be lost or gained more frequently than others (Figs. 2 and 3). Losses of normal homologues were noted in all cases and were m u c h more frequent than chromosome gains. Overall (in seven of 11 and six of 11 cases, respectively), chromosomes 9 and 19 were lost most often, followed by losses of chromosomes 15, 21, 5, 11, 13, 17, 18, and 22. In three of 11 breast carcinoma samples, a clonal loss could be detected (MaCa 40, #20; MaCa 47, #17; MaCa 85, 21; Table 4]. A loss of chromosome 17 was also observed in two of five aneuploid cells in MaCa 41. The chromosome m a i n l y involved in gains, according to frequency, was #7. The normal homologue of chromosome 18 was gained in only one patient, in w h o m the gain was clonal (MaCa 40; Table 4). Chromosomes 1 and 14 were never lost, and chromosomes 4, 5, 11, 14, and 20 were never gained (Fig. 2 and 3). Structural chromosomal abnormalities could be identified in nine of the analyzed

224

D. G e l e i c k et al.

Table 4

C h r o m o s o m e a n a l y s e s of t u m o r - d e r i v e d c u l t u r e s

Case

Days in culture

Total cells

Normal cells

Pseudodiploid cells

Aneuploid cells

MaCa 30 MaCa 33 MaCa 33

21 21 25

22 13 12

18 9 7

1 1 2

3 3 3

MaCa 34

25

25

18

2

5(1)

MaCa 39 MaCa 39

10 19

11 18

5 16

0 0

6(1] 2

MaCa 40

12

40

23

3

14

No. and karyotypes with identified structural abnormalities including clonal numerical changes o 1 1 I 1 1 1 1 1 2 1 2 1 1 I l 1 1

MaCa MaCa MaCa MaCa

41 47 57 85

12 25 14 16

12 19 14 15

4 9 7 5

3(1) 0 3[4) 3(3}

5 10 4(4) 7(1)

MaCa 86 MaCa 89

15 15

11 4

7 3

1(1) 1

3(3) 0

3 1 1 1 1 0 (I

46.XX,7q + 46,XX,t(6;12}(p21;p13] 46,XX,7q + ,t[6;12}[p21;p13) 46.XX,?inv{5) 47,XX,+inv(7)(q112q32} 47,XX, + iuv(7)(qll 2q32), del(9)(q13) 47,XX, + inv(7}{qll.2;q32) 49,X, ? X , + 8 , + 9 , + i n v ( 7 } (qll.2;q32} 45,XX,- 20 46,XX,+ 7, 20 47,XX, + 18 47,XXX,- 15, + 18 51,XX,+ 1 , + 2 , + 13, + 15, + 18 44,XX, 9, 18,7p46,XX,t[1;9)(p13;p13), t(14,22)(q13;pl 1) 46,XX,del[13)(q14) 46,XX,- 22,del(5){q31.2}, del{14)(q23), + M1 45,XX, - 17 47,XX,+ 16,9p 44,XX, 7, 21 44,XX,- 1 9 . - 21 45,XX, 21

Numbers in parentheses are the total number of unidentified cbromosonms of all karyotyped cells. t u m o r - d e r i v e d c u l t u r e s . K a r y o t y p i n g of c e l l s f r o m t h e c a s e s w i t h a m o d e of 46 c h r o m o s o m e s s h o w e d p s e u d o d i p l o i d cells in n i n e of 11 t u m o r - d e r i v e d s p e c i m e n s ( e x c e p t i o n M a C a 39 a n d M a C a 47). Of 151 a n a l y z e d t u m o r cells w i t h 46 c h r o m o s o m e s , 20 (13.2%) s h o w e d p s e u d o p l o i d i s m r a n g i n g f r o m 0% (MaCa 39, M a C a 47) to 4 3 % (MaCa 41; T a b l e 4). N i n e of 11 t u m o r s p e c i m e n s c o n t a i n e d s t r u c t u r a l l y a b n o r m a l c h r o m o s o m e s . M o s t of t h e m a r k e r s w e r e e x t r e m e l y c o m p l e x a n d c o u l d be i d e n t i f i e d c o m p l e t e l y o n l y to a s l i g h t e x t e n t ( T a b l e 4). T w o t r a n s l o c a t i o n s in M a C a 40 [t(1;9) a n d t(14;22)] a n d d e l e t i o n s of c h r o m o s o m e 5 (MaCa 41), 9 (MaCa 39), 13 (MaCa 40), a n d 14 (MaCa 41) c o u l d be d e t e c t e d . In t w o c a s e s a d e c r e a s e in l e n g t h of c h r o m o s o n m s w a s f o u n d (MaCa 40, # 7 ; M a C a 57, # 9 ) . C l o n a l s t r u c t u r a l c h a n g e s w e r e rare. A p r i m a r y b r e a s t c a n c e r M a C a 33, f r o m a 50y e a r - o l d w o m a n w i t h a n i n v a s i v e d u c t a l c a r c i n o m a , s h o w e d a c l o n a l t r a n s l o c a t i o n of c h r o m o s o m e 6 w i t h 12 (Fig. 4) a n d a n e n l a r g e d c h r o m o s o m e 7. Case M a C a 39, a 43year-old woman diagnosed with invasive ductal carcinoma, showed a clonal inversion of c h r o m o s o m e 7 ( i n v ( 7 ) ( q 1 1 . 2 q 3 2 ) ; Fig. 5). T h e s a m e s t r u c t u r a l a b n o r m a l i t y c o u l d be v e r i f i e d i n M a C a 34 (a 4 6 - y e a r - o l d w o m a n w i t h i n v a s i v e d u c t a l c a r c i n o m a ) in o n e cell (Fig. 6). In n o n e of t h e a n a l y z e d m e t a p h a s e s w e r e d m i n or H S R f o u n d .

22 5

Cytogenetics of Breast Cancer

< *

I:

1

2

3

6

7

8

13

14

15

19

20

21

Figure 4

9

22

4

5

10

11

12

16

17

18

XX

Karyotype of MaCa 33 (25 days ill culturel 46,XX,t(6;12](p21;p13).

DISCUSSION Tumor-derived cultures could be distinguished from cultures of normal breast epithelial cells by their m o d a l distribution curves (Fig. 1). Cells in the tetraploid range could be found in both culture types, but a p r o n o u n c e d h y p e r d i p l o i d y and an increased frequency of h y p o d i p l o i d cells was a characteristic feature of tumor-derived cultures. In the present study, these differences were statistically significant. In addition, we identified clonal losses and gains, in contrast to results of a recent study by Zhang et al. [12] in w h i c h numerical c h r o m o s o m a l aberrations a p p e a r e d to be r a n d o m and nonclonal. In breast cancer, n o n r a n d o m involvements of c h r o m o s o m e s 1p(1p11-21), lq(1223), 3p, 6q, 8, 11p, 11q(11q13-24), 13q, 16q and 17 have been reported (for review, see refs. 3 and 8). We were able to demonstrate clonal structural abnormalities in two primary breast cancers. A clonal translocation between c h r o m o s o m e s 6 and 12 (p21;p13) and an enlarged c h r o m o s o m e 7 (7q+) in a p r e m e n o p a u s a l 50-year-old patient with invasive ductal carcinoma was observed. This is the first report of a clonal t(6;12) in breast cancer. Hitherto, losses of 6q [101 and translocations with chromosomes other than c h r o m o s o m e 12 [9] have been observed. The second structural clonal a b n o r m a l i t y inv(7)(q11.2q32) occurred in a 43-year-old hysterectomized w o m a n with invasive ductal carcinoma. Although not clonal, the same genetic change was detected in a 46-year-old p r e m e n o p a u s a l w o m a n also diagnosed with invasive ductal carcinoma. A recent study s h o w e d frequent involvements of c h r o m o s o m e 7, among w h i c h a clonal inversion was also detected (inv(7)(p13q36); [31]). Chromosome 7 was the c h r o m o s o m e most frequently involved in structural changes and was observed to be gained preferentially in our cultures. Similar results were been reported

226

D. Geleick et al.

....

1

2

3

6

7

8

13

14

15

19

20

21

Figure 5

9

22

4

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Karyotype of MaCa 39 [10 days in culture) 47,XX,+inv(7)(q11.2q32),del(9)(q13).

by Ferti-Passantonopoulou et al. [9] and Gebhart et al. [31]. The gene coding for the EGF-R has been assigned to c h r o m o s o m e 7 ( p l l - p 1 3 ) [32]. An inverse correlation between the presence of ER and of EGF-R was found in primary breast cancer. EGFR-positive tumors were reported to be of poor differentiation and associated with poor prognosis [33-35]. EGF-R data were available from only four tumors. Structural alterations observed in this study were not related to the EGF-R locus. There was no clonal gain of chromosome 7. The overall picture of c h r o m o s o m e 7 might represent the early stage of tumor progression. In three tumor samples we found clonal numerical changes. A gain of c h r o m o s o m e 18 could be detected in a 58-year-old p o s t m e n o p a u s a l w o m a n with m e d u l l a r y carcinoma. The significance of this finding remains to be established. Clonal losses of c h r o m o s o m e 20 (MaCa 40), c h r o m o s o m e 17 (MaCa 47), and c h r o m o s o m e 21 (MaCa 85) were detected. The n o n r a n d o m loss of chromosomes in our study might stress the theory of involvement of t u m o r - s u p p r e s s o r genes in tumor growth and malignancy. In breast cancer, loss of heterozygosity as evidenced by RFLP analysis has been demonstrated for chromosomes 11 [13], 13 [14], 17 [15], and 20 [14]. In 7% of primary tumors, structural aberrations at the retinoblastoma locus (RB) have been identified [36]. A 61% loss of heterozygosity of c h r o m o s o m e 17p with no association of prognostic factors was reported recently for breast tumor cases [15]. Two other studies have also demonstrated 17p deletions [10, 37]. In the present study we found a clonal loss of c h r o m o s o m e 17 in one patient. In a second patient the same c h r o m o s o m e was lost in two of five a n e u p l o i d cells. Both patients had local recurrence after 12 and 11 months, respectively. Chromosome 17 recently received special attention owing to

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Figure 6 Karyotype of MaCa 34 (25 days in culture) 47,XX,+ inv{7)(q11.2q32).

the location of p53, w h i c h has been identified as a potential antioncogene (17p13) [38, 39[. In addition, a clonal 3p(14-21) deletion was found in a primary infiltrating ductal carcinoma [12]. The same deletion was also found in small cell lung cancer [40]. A deletion at the RB locus (del(13)(q14)) was identified in this study in one patient. Cytogenetic equivalents of gene amplification (dmin, HSR) have been described for p r i m a r y breast cancer [10, 18]. In this study no evidence for the presence of drain and HSR could be demonstrated, in agreement with the belief that d m i n and HSR, represent late events in t u m o r progression [3]. In another report [7] drain were found in only 2% of all cases and no evidence for the occurrence of HSR was obtained. MaCa 40, a t u m o r negative for both ER and PR, expressed a high level of EGF-R. As already described, absence of ER and an increased level of EGF-R correlated with bad prognosis. Cells of MaCa 40 were characterized by clonal gain of c h r o m o s o m e 18, clonal loss of c h r o m o s o m e 20, deletions, and translocations. MaCa 47, cytogenetically characterized by a clonal loss of c h r o m o s o m e 17, represented one of the tumors with a local recurrence and progressive disease. Case MaCa 39 without node i n v o l v e m e n t but d e v e l o p m e n t of contralateral disease was characterized by a clonal inversion in c h r o m o s o m e 7. In MaCa 41, w h i c h also showed local recurrence, no clonal aberrations could be identified in the karyotypes analyzed. Further correlations between clinical data and cytogenetic findings appear impossible at the present stage. A follow-up of i n d i v i d u a l patients in c o m b i n a t i o n with a classic and molecular cytogenetic a p p r o a c h [13, 14, 36] s h o u l d increase the possibility of associating t u m o r progression with cytogenetic events.

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This study was partially supported by Swiss National Science Foundation Grant No :1.818.0.84 to Hj.M. ER, PR, and EGF-R data were provided by Professor U. Eppenberger and Dr. D. Fabbro [Research Department, University of Basel, Switzerland). Tbe excellent technical assistance of Mrs. C. Schluchter and Mrs. A. Panebianco (Children's Hospital of Basel) is acknowledged. Dr. K. Tanner performed the statistical analysis. We are grateful to Dr. C. Turc-Carel, Laboratory of Cytogenetics, Dijon, France,, for support in interpreting the cytogenetic data and most valuable discussions.

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