Interphase cytogenetics of a male breast cancer

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  • 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%.

    INTRODUCTION

    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 limited 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-specific probes. In addition, simultaneous analysis of tumor labeling index by in vivo bromodeoxyuri- dine (BrdU) incorporation allowed the proliferation rates of cytogenetically different subpopulations 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.

    243

    ~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 examination 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, binding 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 simultaneously 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 within 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 predominantly in four copies per cell, chromosomes 11, 15, and 18 were present predominantly in three copies per cell, and chromosome 9 was present at two copies per cell. Note that for all chromosomes

  • Cytogenet ics of Male Breast Cancer 245

    Table 2 FISH of in terphase male lymphocytes

    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

    Totalnumber: 178 333 497 342 299 242 297 381 383

    ana lyzed except for chromosome 9, the d is t r ibut ions of copy number were heteroge- neous showing s igni f icant minor subpopu la t ions . A l though the largest subpopu la t ion of cells had four cent romer ic copies for chromosomes 1, 7, and 17, s igni f icant numbers of nuc le i were also seen w i th three and two copies. S imi lar ly , chromosomes 11, 15, and 18 had the largest number of nuc le i w i th three copies, but also had a large propor t ion w i th two copies All of these chromosomes had less than 10% of nuc le i w i th one per icent romer ic copy.

    Both sex chromosomes also showed abnormal d is t r ibut ions . The X chromosome was present most ly in two copies (84.7%); for the Y chromosome there was a signifi- cant minor (12%) popu lat ion , w i th two copies but no cells lacking the Y chromosome.

    S imul taneous detect ion of BrdU incorporat ion by immunof luorescence and chro- mosome copy by in s itu hybr id i za t ion a l lowed the label ing ind ices of d i f ferent sub- popu la t ions w i th in heterogeneous tumors to be measured directly. No s igni f icant d i f ferences were found in BrdU label ing index among cytogenet ica l ly d i f ferent sub- popu la t ions for all of the chromosomes analyzed. The overal l BrdU label ing index was 13.2%.

    DISCUSSION

    A s igni f icant degree of heterogene i ty was seen in the in terphase cytogenet ic d istr ibu- t ions for the ind iv idua l chromosomes tested. Varying propor t ions of nuc le i had two, three, and four copies of the cent romer ic targets. Some of the d i smnic cells present in an o therwise t r isomic (11, 15, 18) or tetrasomic (1, 7, 17) d i s t r ibut ion might have been due to an admixture of d isomic normal cells (at most 10-15%). A l ternat ive ly , count ing errors might arise due to over lap of cent romer ic s ignals w i th in the nuc leus ,

    Tab le 3 FISH of in terphase male breast cancer cel ls

    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 composition of the tumor.

    Chromosome X showed two copies and chromosome Y predominantly 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 subpopulations 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 subpopulations, is easy and reliable, and can be used in combination with other cellular markers [7]. This case exhibits much greater cytogenetic abnormality 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.

    REFERENCES

    1. Balazs M, Mayall BH, Waldman FM. (1991): Simultaneous analyses of chromosomal aneu- ploidy and bromodeoxyuridine incorporation in the MCF-7 breast tumor cell line. Cancel Genet Cytogenet (in press).

    2. DeVita VT Jr, Hellman S, Rosenberg S (1988): Cancer, Principles and Practice, I.B. Lippincott Company, Philadelphia.

    3. Dutrillaux B, Muleris M. Seureau MG (1986): Imbalance of sex chromosomes, with gain ot early-replicating X, in human solid tumors, lnt J Cancer 38(4]:475-479.

    4. Evans DB, Crichlow RW (1987): Carcinoma of the male breast and Klinefelter's syndrome: is there an association? Ca: Cancer l Clinic 37(4):246-251.

  • Cytogenet ics of Male Breast Cancer 247

    5. Gerbault-Seureau M, Vielh P, Zafrani B, Salmon R, Dutrillaux B (1987): Cytogenetic study of twelve human near-diploid breast cancers with chromosomal changes. Ann Genet 30(3):138-145.

    6. Langlands AO. Maclean N, Ken GR (19761: Carcinoma of the male breast: Report of a series of 88 cases. Clin Radiol 27:21-25.

    7. Lichter P, Ward CD (1990): Is non-isotopic in situ hybridization finally coining of age? Nature 345:93-95.

    8. Ljung B, Mayall HB, Lottich C, et al (1989): Cell dissociation techniques in human breast cancer; variations in tumor cell viability and DNA ploidy. Breast Cancer Res and Treat 13:153-159.

    9. Mitchell E (1990): A cytogenetic study of male breast cancer. Cancer Genet Cytogenet 47(1):107-112.

    10. Pinkel D, Straume T, Gray WJ (1986): Cytogenetic analysis using quantitative, high sensitiv- ity fluorescence hybridization. Proc Natl Acad Sci USA 83:2934-2938.

    11. Rodgers SC, Hill MS. Hulten MA (1985): Cytogenetic analyses in a case of cancer of the male breast. Cancer Genet Cytogenet 15:113-117.

    12. Shackney SE, Smith CA, Miller BW, Burholt DR, Murtha K, Giles HR, Ketterer DM, Police AA (1989): Model for the genetic evolution of human solid tumors. Cancer Res 49:3344-3354.

    13. Waldman MF, Balazs M, Mayall HB, Pinkel D, Gray J (1991): Karyotypic heterogeneity and its relation to labeling index in interphase breast tumor cells. Cancer Res (in press).

    14. Wolman SR, Camuto PM, Perle MA (1988): Cytogenetic diversity in primary human tumors. J Cell Biochem 36:147-156.

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