ELSEVIER

Cytogenetic Report of a Male Breast Cancer

Luciane Regina Cavalli, Silvia Regina Rogatto, Claudia Aparecida Rainho, Marcel0 Jo& dos Santos, Iglenir Joao Cavalli, and Dora Maria Grimaldi

ABSTRACT: The cytogenetic findings on G-banding in an infiltrating ductal breast carcinoma in a 69- year-old man are reported, The main abnormalities observed were trisomy ofchromosomes 8 and 9 and structural rearrangement in the long arm ofchromosome 17 (add(l7)(@5)). Our results confirm the trisomy of chromosome 8 in the characterization of the subtype of ductal breast carcinomas and demonstrate that chromosome 17, which is frequently involved in female breast cancers, is also responsible for the develop- ment or progression of primary breast cancers in males.

INTRODUCIION

Male breast cancer represents approximately 1% of all breast cancers. We are aware of just six cases studied cytogeneti- tally [l-5]. In these reports, the cytogenetic analysis after G-banding treatment revealed karyotypic alterations affect- ing mainly chromosomes X, 3, 5, 6, 7, and 11. We describe a further cytogenetic study of a male breast carcinoma.

CASE REPORT

A 69-year-old man, father of two sons, with no testicular anomalies, was sent to a Health Service due to the appear- ance of a firm and mobile nodule, approximately 3 cm in diameter, localized in the upper inner quadrant of his right breast. After 1 month, a total mastectomy was performed af- ter diagnosis of a well-differentiated invasive ductal carci- noma of tubular pattern in his right breast.

Histologically, the tumor consisted of epithelial neoplas- tic cells, of tubular type, with free margins, anisocitose, anisocoriose, proeminent nucleolis, and with microme- tastases, grade I, in the lymph nodes (Figs. 1 and 2).

One month after the surgery the patient was administered eight cycles of chemotherapy, at intervals of approximately 30 days, during a period of 10 months. Each chemotherapeu- tic cycle was composed of a dose of 800 mg of eduxon, 800

From the Departamento de Genetica, Universidade Federal do Pamnh, Curitiba, Pr (L. R. C., I. J. C.), Departamento de Genetica, UNESP, Botucatu, SP IS. R. A. J. Departamento de Biologia Geml, CCB (C. A. R., M. J. S.) and Departamento de Patologia Aplicada, Legisla@o e Deontologia [D. M. G.), Universidade Estadual de Lon- drina, Londrina, Pr, Brazil.

Address reprint requests to: Luciane Regina Cavalli, Depar- tamento de GenBtica, Universidade Federal do Pamn6, CX 19071 CEP 81531-970, Curitiba, Parand, Bmzil, CX 1907l FAX# 55-41- 2662042

Received March 7, 1994; accepted September 20, 1994.

Cancer Genet Cytogenet 81:66-71 (1995) 0 Elsevier Science Inc., 1995 655 Avenue of the Americas, New York, NY 10010

mg of methotrexate, and 750 mg of 5-fluoracil. The radiother- apeutic treatment occurred 4 months after surgery for 1 month (total dose, 5000~ Gy/D of telecobaltotherapy).

MATERIALS AND METHODS

A sample of fresh and non-treated tumor, an infiltrating duc- tal breast carcinoma, was obtained for cytogenetic analysis from the patient immediately after surgery. The material was sent by the Hospital Universitario de Londrina, South of Brazil.

A sample of 10 ml of peripheral blood was obtained for a constitutional chromosome analysis, 1 month after eight cycles of chemoradiotherapeutic treatment to which the pa- tient was submitted after the surgical intervention.

The tumor tissue fragments sent to the laboratory for chm- mosome evaluation were sectioned after removal of fatty and necrotic areas. A 0.3 % type IV collagenase solution (Sigma) was used for enzymatic dissociation for approximately 20 minutes. The material was incubated at 37°C in Ham F-10 medium (Sigma) supplemented with 20% fetal calf serum, vitamins, and antibiotics. The method used to obtain chro- mosomes was cell synchronization, described by Yunis [6] with modifications. Methotrexate at a final concentration of IO- 7 M was added for 16 hours to block cells in the S phase, and the blockade was released with thymidine at a final con- centration of lo- 5 M for approximately 8 hours. Colchicine (0.0016%) was then added for approximately 15 hours. The standard technique for chromosome preparation was fol- lowed using hypotonic 0.075 M KC1 and methanol:acetic acid (3:1) as fixative. Chromosome studies were made of primary cultures only. The mean time for cultures was 20 days (range, 15-50 days).

Peripheral blood was cultured for 72 hours in RPM1 1640 medium (Sigma) containing 20% fetal calf serum and 0.4% phytohemagglutinin.

0165-4608/95/$9.50 SSDI 0165-4608(94)00194-G

Cytogenetics of a Male Breast Cancer 67

Figure 1 Infiltrating breast carcinoma, intraductal type, with proeminent nucleoli.

The G-banding technique was that of Scheres [7], modi- fied. Chromosomes were identified and classified accord- ing to the nomenclature proposed by the International Sys- tem for Human Cytogenetic Nomenclature (1985) [a]. The ISCN 1991 [9] was used for karyotypic description.

RESULTS

A total of 126 tumoral cells were analysed after standard stain- ing; a diploid modal chromosome number was obtained: 68% of cells with 46 chromosomes, 25% with 47 chromo- somes.

We detected in 25 tumor cells analyzed after G-banding the following clonal chromosome abnormalities: trisomy of chromosome 8 (14 cells), trisomy of chromosome 9 (five cells), and monosomy of chromosomes 12 and 17 (three cells). In 68% of the metaphases analyzed (17 cells), a structural chromosome rearrangement was detected, add(17)(q25) (Figs. 3 and 4). A chromosome marker, not identified, was found in two cells. Only three metaphases with a normal comple- ment were observed in this case.

The composite karyotype of the tumor was the follow- ing: 44-48,XY, +8[14],+9[5],-12[3],-17[3],add(V)(q25)[17], +mar[2][cp25].

Figure 2 Lymph node with a micrometastasis in a subcapsular lymphatic vase.

68 L. R. Cavalli et al.

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6 8 9 10 11 12

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Figure 3 The arrows indicate trisomy of chromosome 8 and the add(U)(q25).

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6 7 8 9 10 11 12

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Figure 4 Cell with a 47,X, -Y, + 8, + %add(V)(qz5) karyotype.

Cytogenetics of a Male Breast Cancer 69

Figure 5 Partial karyotypes showing the presence of dicentric and marker chromosomes observed in the analysis of the peripheral blood.

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Figure 6 Patient karyotype showing a normal chromosomal constitution (46,XY).

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The karyotypic analysis of 26 cells of the peripheral blood, submitted to the G-banding treatment, revealed clonal chro- mosome alterations: monosomy of chromosomes 11,12, 19, and 21 (three cells) and trisomy of chromosome 19 (18 cells). In six cells, an unspecified dicentric chromosome was ob- served (Figs. 5 and 6).

DISCUSSION

The four male breast cancers previously investigated cytoge- netically with banded analysis [l-3] demonstrated a near- diploid chromosome number. In these reports, clonal chromosome abnormalities involved an additional X chro- mosome, monosomy of chromosomes 1, 6, and 8, trisomy of chromosome 7, and structural rearrangements in chromo- somes 1, 6, 11,13, and 14. Mitchell [4], using direct prepara- tion in a study of a male breast cancer, observed the trisomy of chromosome 3, monosomy of chromosome 7, and the in- volvement of chromosomes 5 and 11 in the marker chromo- some formation. The interphase cytogenetic analysis (FISH) performed on nuclei from a male infiltrating ductal breast tumor suggested the presence of polysomy involving chro- mosomes 1, 7, 11, 15, 17, and 18, monosomy of chromosome 9, and one copy of the Y chromosome [5].

The consistent abnormalities found in our study indicate the clonal origin of this tumor. The cytogenetic results sug- gest that the add(l7)(q25) is a primary change and that the trisomies of chromosomes 8 and 9 are secondary changes in the neoplastic process.

Chromosome 17 contains several genes important to the development or progression of breast cancers. Already iden- tified are the TP53 tumor suppressor gene on 17~13.1 [lo, 111 and the ERB-B2 gene on 1Zq [12, 131. Cropp et al. [14] have shown a high frequency of loss of heterozygosity on 17q that was associated with estrogen receptor-negative tumors. Sev- eral groups have published evidence for a gene on li’q linked to familial breast/ovarian cancers [15, 161. The gene, named BRCA 1, maps to lip21. Additional chromosome material localized at 17q25, as described in the present study, was not observed in the previous reports. The region between ljp25 and lip25.3 was identified as one of the two regions com- monly deleted in breast tumors [%‘I. Near the ljp25 region, the sites for thymidine kinase proteins (TKl) and for growth hormones (GHl and GH2) [18] were mapped. The long arm of chromosome 17 is considered to contain the gene respon- sible for hereditary forms of breast and ovarian cancers and is considered to contain one or more genes that play a sig- nificant role during the development or progression of pri- mary cancer in both of these tissues [w]. In male breast tumors, Labaccaro et al. [18] studied the possible role of the androgen receptor (AR) gene in the development of these tumors and they reported a germline mutation of the AR. The authors suggested that the breast cancer gene, BCRA 1, acts as a tumor supressor with an androgenic inhibition of cell proliferation via increased 17(3-hydroxysteroid oxydative activity (located within the linked interval) in breast tumors.

The trisomy of chromosome 8, observed with high fre- quency (56% of the cells) in this study, was not described in male breast cancer, but it has already been described in leukemias, lymphomas, and in some solid tumors [20]. In

L. R. Cavalli et al.

breast tumors, it was found as a recurrent karyotypic abnor- mality, representing an early genetic change that character- izes a subtype of ductal breast carcinoma [21]. Our results are in agreement with this characterization, due to the high frequency at which the trisomy of this chromosome was ob- served in this type of histologic pathology. Because trisomy 8 occurs in benign as well as malignant tumors, it may en- dow the cells with a growth advantage not necessarily related to malignancy but dependent on the presence of cytogeneti- tally visible or invisible mutations [21].

The trisomy of chromosome 6 was also not observed in male breast cancer. In female breast tumors there is only one report describing the involvement of this chromosome (in the 6q12 region) in structural rearrangements [22].

Clinical conditions that result in hypoandmgenism, such as Klinefelter syndrome [23-251, testicular atrophy, orchitis, undescended testes, testicular trauma, and infertility, have been determined to appear to put men at increased risk for breast cancer [lg]. Considering this information, we per- formed the chromosome analysis of the peripheral blood, observing a normal karyotype (46,XY). However, this analy- sis revealed chromosome abnormalities (involving chromo- somes 11,12,18, and 21, as well as the presence of dicentric and markers chromosomes) that, in spite of being clonal, were distinct from those observed in the tumoral cells and there- fore cannot be involved in the tumorigenic process. The pres- ence of these clonal abnormalities in the peripheral blood, especially the trisomy of chromosome 16, which is frequent in ANLL, could suggest a hematologic malignancy in the patient. However, the clinical follow-up of the patient for 28 months did not demonstrate any evidence of a hematologic disease, suggesting that these abnormalities are probably the results of the chemoradiotherapeutic treatment administered to the patient.

The authors thank the staff of the Gynecology and Pathology Depart- ment, Hospital Universitario de Londrina, Pr, Brasil, and CPG- Universidade Estadual de Londrina, CAPES, CNPq (National Re- search Council for Scientific and Technologic Development).

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