Interphase Cytogenetics of a Male Breast Cancer

Margit Balazs, Brian H. Mayall, and Frederic M. Waldman

ABSTRACT: Direct interphase cytogenetic analysis was performed on nuclei from a male breast tumor using fluorescence in situ hybridization (FISH). DNA probes specific for repetitive pericentro- meric regions on chromosomes 1, 7, 9, 11, 15, 17, 18, X, and Y were used to determine chromo- some copy numbers in interphase tumor cells. Copy number distributions varied greatly between chromosomes, showing major tumor populations with one (Y). two (X, 9), three (11, 15, 18), and four (1, 7, 17) copies of the pericentromeric targets. The X chromosome was present in two copies in 84.7% of tumor nuclei, with the balance being primarily monosomic. Normal skin fibroblasts cultured from the same patient showed 99% monosomy X. The Y chromosome showed a minor population (12%) with two copies. The DNA index of the tumor was 2.0 as determined by flow cytometry. The proliferative activity of the tumor cells was simultaneously analyzed using detection of in vivo bromodeoxyuridine (BrdU) incorporation. The BrdU labeling index was 13.2%.

I N T R O D U C T I O N

Cancer of the breast in males is a rare disease with an incidence of only 1% compared with that in women [2]. The tumor is often less well defined in men than in women, and because of l imited breast tissue it may be closely applied to pectoral fascia and can involve the muscle itself [2]. Although stage for stage prognosis is similar for men and women, male breast cancer frequently presents at a more advanced stage [6].

Cytogenetic analysis using chromosome banding techniques have been reported for only 5 male breast cancers (Table 1). Although rare families in which several males have developed breast cancer have been reported and Klinefelter (XXY) males may have an increased incidence of breast cancer [4], no common numerical or structural chromosome aberrations have been found in the cases analyzed by chromosome banding techniques. Rather, the cytogenetic findings were similar to the range of defects seen in breast cancers in women [5, 14].

In this study we further characterize the cytogenetics of male breast cancer by describing the first karyotypic analysis of a male breast cancer using fluorescence in situ hybridization (FISH) [7, 10] with nine different chromosome-specif ic probes. In addition, s imultaneous analysis of tumor labeling index by in vivo bromodeoxyuri- dine (BrdU) incorporation al lowed the proliferation rates of cytogenetically different subpopulat ions to be measured.

From the Department of Laboratory Medicine. University of California San Francisco, San Francisco. California.

Address requests for reprints to: Frederic Waldman, Bax 0808. University of CaliJbrnia San Francisco, San Francisco, CA. 94143-0808.

Received December 28, 1990; accepted February 11, 1991.

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~3 1991 Elsevier Science Publishing Co., Inc. Cancer Genet Cytogenet 55:243- 247 {1991) 655 Avenue of the Americas, New York, NY lt)010 0165-4608/91/$03.50

244 M. Balazs et al.

Table 1 Cytogenetic studies in reported male breast cancers

Number of metaphases Normal Reference

Case Aberrations detected analyzed karyotype number

1 47,XXY 3 46,XY 5 46,XY 4

2 46,XY 12 46,XY 5 Trisomy lq, Monosomy and trisomy 6, Monosomies 1, 11, and 13

3 47,XXY (cell line) 46,XY 3 4 34-48, XY 73 46,XY 11

Numerous chromosomal losses, 79.XY 46,XY

5 Hypodiploid (25-34) 2 46,XY 9 Hyperdiploid (56-84) 11

MATERIALS AND METHODS

The patient was a 35-year-old man presenting with a 2.2-cm mass in the left breast. He gave informed consent for in vivo BrdU administration, and was given 200 mg/ m 2 BrdU intravenously during the 30 minutes prior to surgery. Breast tumor and skin biopsy samples were obtained immediately following tumor excision [8]. Histologic examinat ion showed poorly differentiated infiltrating ductal carcinoma; 28 of 28 axillary lymph nodes were free of tumor. Single-cell suspensions from fresh tumor material were obtained by mechanical dissociation and Carnoy's fixation as described [1,8]. Cultured human male lymphocytes from healthy donors were used as hybridiza- tion controls. DNA probes used were specific for mostly c~-satellite repetitive se- quences on individual chromosomes, b inding to peri-centromeric regions [1]. Probes were labeled with biotin by nick translation using commercially available kits (Bethesda Research Laboratories). FISH was carried out on slides as described by Pinkel et al. [10] with modifications [1]. Two hundred to 600 interphase nuclei were counted for each chromosome analyzed. Flow cytometry was performed as previously described [1]. Tumor DNA labeling index was measured by immunofluorescent detec- tion of BrdU incorporation s imultaneously with FISH [1, 13].

RESULTS

Chromosome copy numbers were defined by FISH with repetitive probes specific for chromosomes 1, 7, 9, 11, 15, 17, 18, X, and Y. The number of fluorescent hybridization signals seen wi thin each nucleus was counted as the number of separate copies of that peri-centromeric sequence present in each cell.

The interphase cytogenetic results for control male lymphocytes are shown in Table 2. Note that more than 95% of nuclei had one copy of the X and Y chromosomes and two copies of all others tested. Also, more than 99% of cultured normal skin fibroblasts obtained from the same patient had 1 copy of chromosomes X and Y and 2 copies for chromosomes 17 and 1 (data not shown).

The FISH results from hybridizations on interphase tumor nuclei are shown in Table 3. Chromosomes 1, 7, and 17 were present predominant ly in four copies per cell, chromosomes 11, 15, and 18 were present predominant ly in three copies per cell, and chromosome 9 was present at two copies per cell. Note that for all chromosomes

C y t o g e n e t i c s of Ma le Breas t C a n c e r 245

T a b l e 2 FISH of i n t e r p h a s e m a l e l y m p h o c y t e s

Chromosome (%)

Signals/cell 1 7 9 11 15 17 18 Y X

0 0 0 0 0.3 1.0 0 0 0.5 0.3 1 1.7 2.1 4.2 2.3 2.3 3.7 2.0 98.7 99.2 2 97.2 97.6 94.8 95.3 95.7 94.6 96.0 0.5 0.5 3 1.1 0.3 0.4 0.9 0.7 1.2 0.7 0.3 0 4 0 0 0.6 1.2 0.3 0.4 1.3 0 0

Tota lnumber : 178 333 497 342 299 242 297 381 383

a n a l y z e d e x c e p t for c h r o m o s o m e 9, the d i s t r i b u t i o n s of copy n u m b e r we re he te roge- n e o u s s h o w i n g s ign i f i can t m i n o r s u b p o p u l a t i o n s . A l t h o u g h the largest s u b p o p u l a t i o n of cel ls h a d four c e n t r o m e r i c c o p i e s for c h r o m o s o m e s 1, 7, a n d 17, s ign i f i can t n u m b e r s of n u c l e i we re a lso s een w i t h t h r ee a n d two copies . S imi l a r ly , c h r o m o s o m e s 11, 15, a n d 18 h a d the la rges t n u m b e r of n u c l e i w i t h t h r ee copies , bu t a lso h a d a large p r o p o r t i o n w i t h t w o copies• All of t h e s e c h r o m o s o m e s h a d less t h a n 10% of n u c l e i w i t h one p e r i c e n t r o m e r i c copy.

• Bo th sex c h r o m o s o m e s also s h o w e d a b n o r m a l d i s t r i b u t i o n s . T h e X c h r o m o s o m e was p r e s e n t m o s t l y in two cop ie s (84.7%); for the Y c h r o m o s o m e the re was a s ignif i - can t m i n o r (12%) p o p u l a t i o n , w i t h two c o p i e s bu t no cel ls l ack ing the Y c h r o m o s o m e .

S i m u l t a n e o u s d e t e c t i o n of B r d U i n c o r p o r a t i o n by i m m u n o f l u o r e s c e n c e a n d chro- m o s o m e c o p y by in s i tu h y b r i d i z a t i o n a l l o w e d the l abe l i ng i n d i c e s of d i f f e ren t sub- p o p u l a t i o n s w i t h i n h e t e r o g e n e o u s t u m o r s to be m e a s u r e d d i rec t ly . No s ign i f i can t d i f f e r ences were f o u n d in B r d U l abe l i ng i n d e x a m o n g c y t o g e n e t i c a l l y d i f f e ren t sub- p o p u l a t i o n s for all of the c h r o m o s o m e s ana lyzed . The overa l l B r d U l a b e l i n g i n d e x was 13.2%.

DISCUSSION

A s ign i f i can t degree of h e t e r o g e n e i t y was s e e n in the i n t e r p h a s e c y t o g e n e t i c d i s t r ibu - t i ons for the i n d i v i d u a l c h r o m o s o m e s tes ted . Vary ing p r o p o r t i o n s of n u c l e i h a d two, th ree , a n d four cop ie s of the c e n t r o m e r i c targets . S o m e of the d i s m n i c cel ls p r e s e n t in an o t h e r w i s e t r i s o m i c (11, 15, 18) or t e t r a s o m i c (1, 7, 17) d i s t r i b u t i o n m i g h t have b e e n d u e to a n a d m i x t u r e of d i s o m i c n o r m a l cel ls (at mos t 1 0 - 1 5 % ) . A l t e r n a t i v e l y , c o u n t i n g e r ro rs m i g h t ar ise d u e to ove r l ap of c e n t r o m e r i c s igna l s w i t h i n the n u c l e u s ,

T a b l e 3 FISH of i n t e r p h a s e m a l e b reas t c a n c e r ce l l s

Chromosome (%)

Signals/cell 1 7 9 11 15 17 18 Y X

0 0 0 0.5 0 2.6 0 0 0 0.3 1 1.6 0 7.0 0.6 6.0 2.3 1.3 87.9 13.5 2 15.4 15.2 91.2 34.1 31.6 14.1 23.7 12.0 84.7 3 34.0 11.6 1.5 61.1 39.4 27.9 65.2 0.04 1.3 4 40.9 73.2 0 4.2 20.0 55.6 9.8 0 0.2 5 0 0 0 0 0.5 0 0 0 0

Total number: 247 310 340 167 425 559 316 249 667

246 M. Balazs et al.

causing four signals to appear as three, or three as two. Based on the number of normal disomic nuclei showing only one signal, overlap error should remain small but will increase with increasing signal number. Thus, the minor populations are more likely due to a true genetic heterogeneity within this tumor.

One possible explanation for the heterogeneous polysomy is that a spontaneous doubling (tetraploidization) of chromosome number per cell occurred at some time during the tumor's evolution [121. Then, with no selective pressure (increased prolifer- ative rate) associated with this genotype, chromosomes were randomly lost, leading to the varied distributions for each of the chromosomes tested. The presence of only one Y chromosome and only two chromosomes 9 might be due to a selective advantage or perhaps due to these chromosomes being excluded during tetraploidization. A significant number of nuclei had two copies of the Y chromosome, suggesting that it too had been present in two copies, but the clone with only one copy became domi- nant. The DNA index of this tumor was 2.0 by flow cytometric analysis, which indicates tetraploidy. The FISH results, showing both tetraploid and hypotetraploid tumor populations, show more heterogeneity than the flow analysis, and perhaps better represent the true cytogenetic composit ion of the tumor.

Chromosome X showed two copies and chromosome Y predominant ly one copy. Imbalance of sex chromosomes was reported by Dutrillaux et al. in several solid tumor cell lines including a male breast carcinoma [3]. In one derived cell line an excess of chromosome X was the major anomaly observed in addition to random nonclonal losses of various other chromosomes. A slight increase in the incidence of breast cancer in males with Klinefelter's syndrome (47, XXY) has been reported [4]. The patient reported in the present study, however, had a normal 46,XY karyotype as determined by FISH of his normal skin fibroblasts.

The in vivo BrdU labeling index of 13.2% is considerably higher than the mean BrdU labeling index of 7.6% seen in 56 women with breast cancers labeled by in vivo BrdU incorporation; only 21% of female breast cancers have a labeling index equal to or greater than 13.2% (unpublished results). It is significant that there was no difference in the BrdU labeling index among the various cytogenetically defined subpopulat ions of the tumor. This implies that none of the cytogenetic variations had a proliferative advantage relative to each other.

In summary, the case reported here exemplifies the advantages of FISH for in- terphase cytogenetics: it is nonselective, allows sufficient ceils to be analyzed to detect minor subpopulat ions, is easy and reliable, and can be used in combinat ion with other cellular markers [7]. This case exhibits much greater cytogenetic abnormali ty than has been reported previously by conventional cytogenetic analyses of male breast cancers.

This work was supported by NCI grants CA 49056, CA 44768, and CA45919. The authors wish to thank Sandra DeVries for her enthusiastic technical support and Dr.

Carrie Gotkowitz for her helpful review of the manuscript.

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