- Email: [email protected]
Clonal chromosomal alterations in fibroadenomas of the breast
Cancer Genetics and Cytogenetics 131 (2001) 120–124
Clonal chromosomal alterations in fibroadenomas of the breast Luciane Regina Cavallia, Déborah Afonso Cornélioa, Lismeri Wuicika, Ana Teresa Schmid Brasa, Enilze M.S.F. Ribeiroa, Rubens Silveira Limac, Cícero Andrade Urbanc, Silvia Regina Rogattob, Iglenir João Cavallia,* a
Departamento de Genética, Universidade Federal do Paraná, Caixa Postal 19071, CEP 81531-970, Curitiba, Paraná, Brazil b Departamento de Genética, IB-UNESP-Botucatu, SP, Brazil c Departamento de Oncologia do Hospital Nossa Senhora das Graças, Curitiba, Paraná, Brazil Received 22 November 2000; received in revised form 26 April 2001; accepted 27 April 2001
Abstract
A cytogenetic study on short-term cell cultures from 10 fibroadenomas of the breast is reported. Clonal chromosomal alterations were observed in all cases analyzed, involving preferentially chromosomes X, 12, 14, 20, and 22. Normal karyotypes were found in 34.9% of the cells. The present findings are discussed together with the reports on fibroadenomas and other benign lesions of the breast described in the literature. Although no specific chromosome abnormality to date can be attributed to a particular type of benign breast pathology, some recurrent alterations are starting to emerge and may characterize these benign breast lesions, differentiating them from their malignant counterparts. © 2001 Elsevier Science Inc. All rights reserved.
1. Introduction Fibroadenoma of the breast is a benign proliferative disease occurring preferentially in women aged 20 to 35 years [1]. It presents as a well- demarcated and mobile tumor that is histologically composed of both epithelial and stromal components [2]. Clonality studies have indicated a polyclonal origin in both types of cells of these tumors [3]; however, monoclonality in cells of stromal origin was found and may indicate these hyperproliferative disorders as neoplastic [4,5]. Molecular studies suggest that the recurrent and monoclonal fibroadenomas (of stromal cells) can progress to phyllodes tumor, a neoplasm of stromal cells [6]. Malignant transformation of the epithelial component however, has been reported in only 0.02–0.3% of the cases [7]. Cytogenetic studies in fibroadenomas of the breast have been described [4,5,8–18], but no specific abnormality has been reported and a large number of tumors analyzed presented with normal karyotypes. Chromosomes involved in the cases with clonal chromosomal alterations include 1, 4, 6, 12, and 20 in structural rearrangements and 5, 11, 17, and 18 in the numerical changes. Although some of these alterations are recurrent in fibroadenomas, they are not specific
* Corresponding author. Tel.: 55-41-361-1728; fax: 55-41-266-1042. E-mail address: [email protected] (I.J. Cavalli).
for these lesions, as many have also been detected in other benign histologic entities, as well as, in breast cancer specimens. In this study, we report clonal chromosomal alterations in 10 fibroadenomas of the breast and discuss the results together with the reports on fibroadenomas and other benign lesions of the breast described in the literature. 2. Material and methods Ten fibroadenomas of the breast were studied cytogenetically following 10–15 days of primary in vitro culture. The samples were obtained from tumors prior to any treatment and were collected at the Hospital Nossa Senhora das Graças, Curitiba, Paraná, Brazil. The clinical data on the patients are presented in Table 1. Primary cultures were started according to standard procedures. Briefly, tumor tissues were minced after removal of fatty areas, and enzymatically dissociated with a 0.3% type IV collagenase solution (Sigma) for approximately 20 min. Cells were cultured in HAM F-10 medium supplemented with 20% fetal calf serum, vitamins, and antibiotics. The cultures were inspected daily and harvested using the method described by Yunis [19] with modifications. Chromosomes were GTG banded [20] and classified following the nomenclature proposed by An International System for Human Cytogenetics Nomenclature (ISCN) [21].
0165-4608/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S0165-4608(01)00 5 0 2 - 7
L.R. Cavalli et al. / Cancer Genetics and Cytogenetics 131 (2001) 120–124 Table 1 Clinicopathologic data of the 10 patients with fibroadenomas analyzed in this study
Case
Age
Menopausal status
1 2 3 4 5 6 7 8 9 10
34 31 29 68 26 37 25 28 38 52
Pre Pre Pre Post Pre Pre Pre Pre Pre Post
Tumor size (cm)
Clinical follow-up (years)a
2.3 2.2 2 3 2.5 2 654 5 3 2.5 5 3.5 3.5 2 2.5 2 3.5 2.5 2.5 4 3.5 2 4 3.5 2 3 2.5 2.3
10 9 10 10 8 10 5 5 5 5
a None of these patients showed any sign of recurrent disease after the clinical follow-up years.
3. Results Normal karyotypes were observed in all the cases analyzed, with frequencies ranging from 7% (case 3) to 77% (case 4). Numerical clonal chromosomal alterations were observed in all but one case (case 9) and structural clonal chromosomal alterations were observed in 60% of the cases. The chromosomes preferentially involved in numerical alterations were 22 (cases 1, 2, 6, 7, and 10), X (cases 2, 3, 5, and 6), 14 (cases 1, 3, 8, and 10), 20 (cases 4, 5, 6, and 10), and 21 (cases 1, 5, and 6), and in structural alterations were chromosomes 6 (cases 5 and 6), 12 (cases 2 and 5), 9 (case 8), 11 (case 6), 13 (case 9), 14 (case 5), 20 (case 2), and X (case 10). Markers chromosomes were observed in cases 3 and 5 in 35.7% and 8.7% of the cells, respectively. The karyotypes obtained in each case are presented in Table 2. Fig. 1 presents the chromosomes involved in the clonal chromosomal alterations distributed per number of cases, and Fig. 2 presents some of the structural chromosomal abnormalities observed in this study. 4. Discussion Current cytogenetic data on fibroadenomas of the breast are heterogeneous and quite limited [4,5,8–18]. Although
121
some recurrent abnormalities have been observed, no specific alteration has been characterized. Additionally, a large number of cases have shown the presence of a high number of normal cells and a diploid distribution DNA [22]. According to Petersson et al. [16] this variability and lack of recurrent abnormalities can be due to the biphasic nature of these tumors and to the different methods of culture utilized in studies. It was demonstrated that clonal chromosomal alterations in fibroadenomas were restricted to the mesenchymal cells, and epithelial cells from the same tumor were cytogenetically normal [4,5,14]. However, clonal chromosomal alterations were also found in fibroadenoma cultures enriched for epithelial cells [13,16]. Biran et al. [23] found that different tumors require different enzyme exposure times for dissociation of epithelial cells from the stromal elements. The addition of serum to a growth medium facilitates the growth of fibroblasts but may inhibit epithelial growth. Indeed, the limited use of serum in culture has been shown not to cause massive proliferation of fibroblast, but clearly to improve the epithelial cell growth [24]. In this study, normal cells were observed in all cases analyzed, with frequencies ranging from 7% to 77.7%. In most of the cases described in the literature, the frequencies of normal cells are higher than the ones observed in some of our cases. Although this can be related to the number of cells analyzed after G banding and while some of the abnormalities observed may not be representative of the in vivo situation, the biphasic nature of these tumors and the different methods of culture utilized in these studies, as mentioned, can account for this variability. It is notable, however, that the modal chromosome number was diploid for all cases analyzed in this report (data not shown). Cytogenetic studies on characterized epithelial and stromal cells of fibroadenomas are important in order to confirm and to understand the implication of the presence of karyotypically normal cells in these tumors. Clonal chromosomal alterations were observed in all cases of fibroadenomas analyzed in this study. The numerical changes involved preferentially chromosomes 22, X, 14, and 20 and the structural changes involved the chromosomes 6, 9, 11, 12, 13, 14, 20, and X. Although a high heterogeneity of chromosome alterations were observed in our study and we cannot rule out the possibility that the time in
Table 2 Karyotypes obtained in the ten cases of fibroadenomas of the breast analyzed after GTG-banding Case
Karyotype
01 02 03 04 05 06 07 08 09 10
4248, XX, 14, 21, 22 [cp10]/46,XX[1] 4446, XX, X, 5, 9, add(12)(q21q24), inv(20)(p13q11), 22[cp15]/46, XX[6] 4448, XX, X, 12, 14, mar [cp13]/46,XX[1] 47, XX, 20[2]/46,XX[7] 4147, XX, X, 6, del(6)(q23), del(6)(q23), t(12;14)(p14;q32), 17, 20, 21, mar [cp21]/46, XX [2] 4245, XX, X, del(6)(q23), i(11)(p10), 12, 17, 18, 19, 20, 21,21, 22 [cp23]/46, XX[2] 4246, XX, 17, 18, 18, 19, 22 [cp22]/46,XX [9] 4346, XX, 9, 10, 14 [cp12]/46, XX, del(9)(q23)[4]/46, XX[18] 46, XX, del(13)(q22)[4]/46, XX[11] 4246, XX, 14, 18, 20, 22 [cp15]/46, X, del(X)(q26)[4]/46, XX[21]
122
L.R. Cavalli et al. / Cancer Genetics and Cytogenetics 131 (2001) 120–124
Fig. 1. Chromosomes involved in clonal chromosomal alterations distributed per number of cases (total of 10 cases analyzed).
culture and/or the tissue culture method used may have favored the preferential growth of specific cells in vitro and, therefore, that some abnormalities observed may not be representative of the in vivo situation, some of the chromosomes involved in the clonal cytogenetic alterations were already described in the literature in other cases of fibroadenomas and benign lesions of the breast, confirming the nonrandom involvement in these benign entities. Monosomy of chromosome 22, the most frequent chromosome loss observed in our study (50% of the cases analyzed), was previously described in fibroadenomas of the breast by Tibiletti et al. [18] and in a mammary fibroepithelial hyperplasia reported by Burbano et al [25]. The monosomy of chromosome X, also observed with high frequency in this report (40% of the cases), was described in other fibroadenomas cases in the literature [18] as well as in a series of breast tumors cytogenetically analyzed [26]. Its significance, however, is unknown and it has been questioned whether they were derived from neoplastic cells [27,28]. Rearrangements on Xq26, observed in case 10, was also observed as the sole structural alteration in one fibroadenoma reported by Petersson et al. [16] as a t(X;1)(q26;p33). Gain of chromosome 20, found in case 4 of this study, was previously reported in other fibroadenomas [4,16,18] and in one case of fibroepithelial hyperplasia of the breast [25]. In case 2, a structural alteration involving chromo-
some 20, inv(20)(p13q11), was observed in 33% of the cells analyzed. This same abnormality was observed in case 4, but as a nonclonal chromosomal alteration. Calabrese et al. [9] reported a fibroadenoma with the involvement of band 20q11 as a structural alteration. Structural alterations involving chromosome 12 have been described with relative frequency in fibroadenomas [9,12,13,15] and other breast diseases [29]. Translocations involving 12p12 were observed by Calabrese et al. [9] in two fibroadenomas. In a phyllodes tumor of the breast, the region 12p12 was deleted [del(12)(p11p12)] [30], and the region 12p13 was translocated [t(3;12)(q21;p13)] [31]. Alterations at 12p12 were also observed in a fibrocystic lesion of the breast [32]. The K-RAS oncogene is localized at 12p12 and is frequently altered in mammary carcinomas [33]. Alterations involving 12q13q15, the site of the oncogenes INT1 and GLI (HGM11, 1991), have been described in subsets of pleomorphic adenomas of the salivary glands, uterine leiomyomas, and lipomas [34] and also in benign epithelial lesions [29,31], including fibroadenomas [9,12,15]. The HGMI-C gene (high mobility group protein) mapped to 12q15 [34], within the region designated MAR (multiple aberration region), was found to be rearranged in several benign mesenchymal neoplasms [35] and in the fibroadenoma described by Staats et al. [15]. In case 2 of this study, additional chromosomal material in band 12q21q24
Fig. 2. Partial karyotypes showing structural chromosomal abnormalities involving chromosomes 6, 11, 12, and 20 (arrows indicate the rearranged chromosome).
L.R. Cavalli et al. / Cancer Genetics and Cytogenetics 131 (2001) 120–124
was observed. Structural chromosomal alterations in chromosome 12 are, therefore, a recurrent and common abnormality in some of the fibroadenomas described in the literature, and further analysis on the breakpoints involved may indicate genes with relevance to fibroadenoma tumorigenesis. Translocations involving chromosomes 12 and 14 were reported by Dietrich et al. [13] in one fibroadenoma, however, the breakpoints were different from the ones observed in the present study. Translocations involving chromosome 14 were also described in fibroadenomas by Belda et al. [5] and Leuschner et al. [11] with the breakpoints affecting bands 14q24 and 14q32, respectively. Rearrangements involving the long arm of chromosome 6, were previously described in fibroadenomas [4,11,18] and in other mammary benign lesions [13,25,33,36]. Tibiletti et al. [18] observed deletions in 6q in 83.9% of the benign tumors analyzed, most of which consisted of fibroadenomas. This same rearrangement was observed with high frequency in premalignant breast lesions, as well as in mammary carcinomas by the same authors, implying that 6q alterations are among the earliest events in the pathogenesis of breast cancer. Several studies in preinvasive and invasive breast tissue have shown loss of heterozygosity at this region, indicating the presence of a tumor suppressor gene [37–39]. A series of genes associated with mammary tumor progression has been mapped at 6q, including growth inhibiting and senescence promoting genes [40]. Rearrangements involving 6q are also common in ovarian carcinomas [41], and allelotyping studies have indicated several distinct loci of allelic imbalance at this region [41,42]. In summary, according to the cytogenetic data available on fibroadenomas and other benign breast lesions in the literature, some recurrent abnormalities, such as structural alterations of 6q or chromosome 12, are starting to emerge. Characterization of these recurrent abnormalities in future studies using molecular analysis may additionally clarify their association with a particular type of benign breast pathology. Finally, although some of the abnormalities described in our study have been described in epithelial malignant breast tumors as well, and may indicate a preneoplastic condition, it is of interest that the clinical follow-up (5–10 years) for all of these patients has indicated no sign of recurrent disease to date. Additional cases of fibroadenomas as well as other biphasic tumors should be investigated in order to understand the meaning of the chromosome alterations observed. The finding of similarity between some chromosome alterations in fibroadenomas and carcinomas of the breast should be considered in an attempt to verify the progression risk (if any) and if it the same genetic pathway is involved in the etiology of both of these tumors. Acknowledgments The authors thank Dr. Bassem Haddad for the careful reading of the manuscript and the staff of the Oncology Service from the Hospital Nossa Senhora das Graças, Curitiba,
123
for providing the samples. This work was partially supported by a research grant from CNPq (National Research Council for Scientific and Technologic Development). References [1] Harris JR, Hellman S, Henderson IC, Kinne DW. Breast Diseases. 2nd Ed. Philadelphia: J.B. Lippincott; 1991. [2] Koerner FC, O’Connell JX. Fibroadenoma: morphological observations and a theory of pathogenesis. Pathol Annual 1994;29:1–19. [3] Noguchi S, Motomura K, Inaji H, Imaoka S, Koyama H. Clonal analysis of fibroadenoma and phyllodes tumor of the breast. Cancer Res 1993;53:4071–4. [4] Fletcher JA, Pinkus GS, Weidner N, Morton CC. Lineage-restricted clonality in biphasic solid tumors. Am J Pathol 1991;138:1199–207. [5] Belda F, Lester SC, Pinkus JK, Pinkus GS, Fletcher JA. Lineagerestricted chromosome translocation in a benign fibrous tumor of the breast. Hum Pathol 1993;24:923–7. [6] Noguchi S, Yokouchi H, Aihara T, Motomura K, Inaji H, Imaoka S, Koyama H. Progression of fibroadenomas to phyllodes tumor demonstrated by clonal analysis. Cancer 1995;76:1779–85. [7] McDivitt RW, Stevens JA, Lee NC, Wingo PA, Rubin GL, Gersell D and the Cancer and Steroid Hormone Study Group. Histologic types of benign breast disease and the risk for breast cancer. Cancer 1992; 69:1408–14. [8] Zhang R, Wiley J, Howard SP, Meisner LF, Gould MN. Rare clonal karyotypic variants in primary cultures of human breast carcinoma. Cancer Res 1989;49:444–9. [9] Calabrese G, Di Virgilio C, Cianchetti E, Franchi PG, Stuppia L, Parruti G, Bianchi PG, Palka G. Chromosome abnormalities in breast fibroadenomas. Genes Chromosom Cancer 1991;3:202–4. [10] Stephenson CF, Davis RI, Moore GE, Sandberg AA. Cytogenetic and fluorescence in situ hybridization analysis of breast fibroadenomas. Cancer Genet Cytogenet 1992;63:32–6. [11] Leuschner E, Meyer-Bolte K, Caselitz J, Bartnitzke S, Bullerdiek J. Fibroadenoma of the breast showing a translocation (8;14), a ring chromosome and two markers involving parts of chromosome 11. Cancer Genet Cytogenet 1994;76:145–7. [12] Ozisik YY, Meloni AM, Stephenson CF, Peier A, Moore GE, Sandberg AA. Chromosome abnormalities in breast fibroadenomas. Cancer Genet Cytogenet 1994;77:125–8. [13] Dietrich CU, Pandis N, Teixeira MR, Bardi G, Gerdes A, Andersen JA, Heim S. Chromosome abnormalities in benign hyperproliferative disorders of epithelial and stromal breast tissue. Int J Cancer 1995;60: 49–53. [14] Rohen C, Staats B, Bonk U, Bartnizke S, Bullerdiek J. Significance of clonal chromosome aberrations in breast fibroadenomas. Cancer Genet Cytogenet 1996;87:152–5. [15] Staats B, Bonk U, Wanschura S, Hanisch P, Schoenmakers EFPM, Van de Vem WJM, Bartnitzke S, Bullerdiek J. A fibroadenoma with a t(4;12)(q27;q15) affecting the HMGI-C gene, a member of the high mobility group protein gene family. Breast Cancer Res Treat 1996; 38:299–303. [16] Petersson C, Pandis N, Rizou H, Mertens F, Dietrich CU, Adeyinka A, Idvall I, Bondeson L, Georgiou G, Ingvar C, Heim S, Mitelman F. Karyotypic abnormalities in fibroadenomas of the breast. Int J Cancer 1997;70:282–6. [17] Dal Cin P, Pauwels P, Moerman P, Qi H, Van Den Berghe H. Hyperdiploidy in benign breast lesions. Cancer Genet Cytogenet 1998;101:162–3. [18] Tibiletti MG, Sessa F, Bernasconi B, Cerutti R, Broggi B, Furlan D, Acquati F, Bianchi M, Russo A, Capella C, Taramelli R. A large 6q deletion is a common cytogenetic alteration in fibroadenomas, premalignant lesions, and carcinomas of the breast. Clin Cancer Res 2000;6:1422–31. [19] Yunis JJ. New chromosome techniques in the study of human neoplasia. Hum Pathol 1981;12:540–9.
124
L.R. Cavalli et al. / Cancer Genetics and Cytogenetics 131 (2001) 120–124
[20] Scheres VMJC. Identification of two Robertsonian translocation with a Giemsa banding technique. Hum Genet 1972;15:253–6. [21] ISCN. An international system for human cytogenetic nomenclature. Mitelman F, editor. Basel: S. Karger, 1995. [22] Ried T, Just KE, Holtgreve-Grez H, duManoir S, Speicher MR, Schröck E, Latham C, Blegen H, Zetterberg A, Cremer T, Auer G. Comparative genomic hybridization of formalin-fixed, paraffinembedded breast tumors reveals different patterns of chromosome gains and losses in fibroadenomas and diploid and aneuploid carcinomas. Cancer Res 1995;55:5415–23. [23] Biran S, Vlodavsky I, Fuks Z, Lijovetzky G, Horowitz AT. Growth of human mammary carcinoma cells from biopsy specimens in serumfree medium on extracellular matrix. Int J Cancer 1986;38:345–54. [24] Pandis N, Heim S, Bardi G, Limon J, Mandahl N, Mitelman F. Improved technique for short-term culture and cytogenetic analysis of human breast cancer. Genes Chromosom Cancer 1992;5:14–20. [25] Burbano RR, Medeiros A, Amorim MIM, Lima EM, Mello A, Barbieri Neto J, Casartelli C. Cytogenetics of epithelial hyperplasias of the human breast. Cancer Genet Cytogenet 2000;119:62–6. [26] Cavalli LR, Cavaliéri LMB, Ribeiro LA, Cavalli IJ, Silveira R, Rogatto SR. Cytogenetic evaluation of 20 primary breast carcinomas. Hereditas 1997;126:261–8. [27] Johansson B, Heim S, Mandahl N, Mertens F, Mitelman F. Trisomy 7 in nonneoplastic cells. Genes Chromosom Cancer 1993;6:199–205. [28] Rogatto SR, Rainho CA, Barbieri-Neto J, Casartelli C. Loss of the sex chromosome in human neoplasias of the nervous system. Rev Bras Genética 1994;17:215–22. [29] Rohen C, Bonk U, Staats B, Bartnitzke S, Bullerdiek J. Two human breast tumors with translocations involving 12q13-15 as the sole cytogenetic abnormality. Cancer Genet Cytogenet 1993;69:68–71. [30] Dietrich CU, Pandis N, Bardi G, Teixeira MR, Soukhikh T, Petersson C, Andersen JA, Heim S. Karyotypic changes in phyllodes tumors of the breast. Cancer Genet Cytogenet 1994;78:200–6. [31] Dietrich CU, Pandis N, Rizou H, Petersson C, Bardi G, Qvist H, Apostolikas N, Boher PJ, Andersen JÁ, Idvall I, Mitelman F, Heim S. Cytogenetic findings in phyllodes tumors of the breast: karyotypic complexity differentiates between malignant and benign tumors. Hum Pathol 1997;28:1379–82.
[32] Nielsen KV, Briand P. Cytogenetics analysis of in vitro karyotype evolution in a cell line established from nonmalignant human mammary epithelium. Cancer Genet Cytogenet 1989;39:103–18. [33] Mars WM, Saunders GF. Chromosomal abnormalities in human breast cancer. Cancer Metastases Rev 1990;9:35–43. [34] Wanschura S, Kazmierczak B, Schoenmakers E, Meyen E, Bartnitzke S, Van de Vem W, Bullerdiek J, Schloot W. Regional fine mapping of the multiple aberration region involved in uterine leiomyoma, lipoma and pleomorphic adenoma of the salivary gland to 12q15. Genes Chromosom Cancer 1995;14:68–70. [35] Schoenmakers EFPM, Wanschura S, Mols R, Bullerdiek J, Van den Berghe H, Van de Ven WJM. Recurrent rearrangements in the high mobility group protein gene, HGMI-C, in benign mesenchymal tumors. Nat Genet 1995;10:436–44. [36] Birdsall SH, Maclennan KA, Gusterson BA. t(6;12)(q23;q13) and t(10;16)(q22;p11) in a phyllodes tumor of breast. Cancer Genet Cytogenet 1992;60:74–7. [37] Theile M, Seitz S, Arnold W, Jandrig B, Frege R, Schlag P, Haensch W, Guski H, Winzer K, Barrett J, Scherneck S. A defined chromosome 6q fragment (at D6S310) harbours a putative tumor supressor gene for breast cancer. Oncogene 1996;13:677–85. [38] Chappel SA, Walsh T, Walker RA, Shaw JA. Loss of heterozygosity at chromosome 6q in preinvasive and early invasive breast carcinomas. Br J Cancer 1997;75:1324–9. [39] Rodriguez C, Causse A, Ursule E, Theillet C. At least five regions of imbalance on 6q in breast tumors, combining losses and gains. Genes Chromosom Cancer 2000;27:76–84. [40] Banga S, Kim S, Hubbard K, Dasgupta T, Jha K, Patsalis P, Hauptschein R, Gamberi B, Dalla Faveera R, Kraemer P, Ozer H. SEN 6, a locus for SV40-mediated immortalization of human cells maps to 6q26-27. Oncogene 1997;14:313–21. [41] Saito S, Saito H, Koi S, Sagae S, Kudo R, Saito J, Noda K, Nakamura Y. Fine-scale deletion mapping of the distal long arm of chromosome 6 in 70 human ovarian cancers. Cancer Res 1992;52:5815–17. [42] Colitti CV, Rodabaugh KJ, Welch WR, Berkowitz RS, Mok SC. A novel 4cM minimal deletion unit on chromosome 6q25.1-q25.2 associated with high grade invasive epithelial ovarian carcinomas. Oncogene 1998;16:555–9.
Clonal chromosomal alterations in fibroadenomas of the breast Luciane Regina Cavallia, Déborah Afonso Cornélioa, Lismeri Wuicika, Ana Teresa Schmid Brasa, Enilze M.S.F. Ribeiroa, Rubens Silveira Limac, Cícero Andrade Urbanc, Silvia Regina Rogattob, Iglenir João Cavallia,* a
Departamento de Genética, Universidade Federal do Paraná, Caixa Postal 19071, CEP 81531-970, Curitiba, Paraná, Brazil b Departamento de Genética, IB-UNESP-Botucatu, SP, Brazil c Departamento de Oncologia do Hospital Nossa Senhora das Graças, Curitiba, Paraná, Brazil Received 22 November 2000; received in revised form 26 April 2001; accepted 27 April 2001
Abstract
A cytogenetic study on short-term cell cultures from 10 fibroadenomas of the breast is reported. Clonal chromosomal alterations were observed in all cases analyzed, involving preferentially chromosomes X, 12, 14, 20, and 22. Normal karyotypes were found in 34.9% of the cells. The present findings are discussed together with the reports on fibroadenomas and other benign lesions of the breast described in the literature. Although no specific chromosome abnormality to date can be attributed to a particular type of benign breast pathology, some recurrent alterations are starting to emerge and may characterize these benign breast lesions, differentiating them from their malignant counterparts. © 2001 Elsevier Science Inc. All rights reserved.
1. Introduction Fibroadenoma of the breast is a benign proliferative disease occurring preferentially in women aged 20 to 35 years [1]. It presents as a well- demarcated and mobile tumor that is histologically composed of both epithelial and stromal components [2]. Clonality studies have indicated a polyclonal origin in both types of cells of these tumors [3]; however, monoclonality in cells of stromal origin was found and may indicate these hyperproliferative disorders as neoplastic [4,5]. Molecular studies suggest that the recurrent and monoclonal fibroadenomas (of stromal cells) can progress to phyllodes tumor, a neoplasm of stromal cells [6]. Malignant transformation of the epithelial component however, has been reported in only 0.02–0.3% of the cases [7]. Cytogenetic studies in fibroadenomas of the breast have been described [4,5,8–18], but no specific abnormality has been reported and a large number of tumors analyzed presented with normal karyotypes. Chromosomes involved in the cases with clonal chromosomal alterations include 1, 4, 6, 12, and 20 in structural rearrangements and 5, 11, 17, and 18 in the numerical changes. Although some of these alterations are recurrent in fibroadenomas, they are not specific
* Corresponding author. Tel.: 55-41-361-1728; fax: 55-41-266-1042. E-mail address: [email protected] (I.J. Cavalli).
for these lesions, as many have also been detected in other benign histologic entities, as well as, in breast cancer specimens. In this study, we report clonal chromosomal alterations in 10 fibroadenomas of the breast and discuss the results together with the reports on fibroadenomas and other benign lesions of the breast described in the literature. 2. Material and methods Ten fibroadenomas of the breast were studied cytogenetically following 10–15 days of primary in vitro culture. The samples were obtained from tumors prior to any treatment and were collected at the Hospital Nossa Senhora das Graças, Curitiba, Paraná, Brazil. The clinical data on the patients are presented in Table 1. Primary cultures were started according to standard procedures. Briefly, tumor tissues were minced after removal of fatty areas, and enzymatically dissociated with a 0.3% type IV collagenase solution (Sigma) for approximately 20 min. Cells were cultured in HAM F-10 medium supplemented with 20% fetal calf serum, vitamins, and antibiotics. The cultures were inspected daily and harvested using the method described by Yunis [19] with modifications. Chromosomes were GTG banded [20] and classified following the nomenclature proposed by An International System for Human Cytogenetics Nomenclature (ISCN) [21].
0165-4608/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S0165-4608(01)00 5 0 2 - 7
L.R. Cavalli et al. / Cancer Genetics and Cytogenetics 131 (2001) 120–124 Table 1 Clinicopathologic data of the 10 patients with fibroadenomas analyzed in this study
Case
Age
Menopausal status
1 2 3 4 5 6 7 8 9 10
34 31 29 68 26 37 25 28 38 52
Pre Pre Pre Post Pre Pre Pre Pre Pre Post
Tumor size (cm)
Clinical follow-up (years)a
2.3 2.2 2 3 2.5 2 654 5 3 2.5 5 3.5 3.5 2 2.5 2 3.5 2.5 2.5 4 3.5 2 4 3.5 2 3 2.5 2.3
10 9 10 10 8 10 5 5 5 5
a None of these patients showed any sign of recurrent disease after the clinical follow-up years.
3. Results Normal karyotypes were observed in all the cases analyzed, with frequencies ranging from 7% (case 3) to 77% (case 4). Numerical clonal chromosomal alterations were observed in all but one case (case 9) and structural clonal chromosomal alterations were observed in 60% of the cases. The chromosomes preferentially involved in numerical alterations were 22 (cases 1, 2, 6, 7, and 10), X (cases 2, 3, 5, and 6), 14 (cases 1, 3, 8, and 10), 20 (cases 4, 5, 6, and 10), and 21 (cases 1, 5, and 6), and in structural alterations were chromosomes 6 (cases 5 and 6), 12 (cases 2 and 5), 9 (case 8), 11 (case 6), 13 (case 9), 14 (case 5), 20 (case 2), and X (case 10). Markers chromosomes were observed in cases 3 and 5 in 35.7% and 8.7% of the cells, respectively. The karyotypes obtained in each case are presented in Table 2. Fig. 1 presents the chromosomes involved in the clonal chromosomal alterations distributed per number of cases, and Fig. 2 presents some of the structural chromosomal abnormalities observed in this study. 4. Discussion Current cytogenetic data on fibroadenomas of the breast are heterogeneous and quite limited [4,5,8–18]. Although
121
some recurrent abnormalities have been observed, no specific alteration has been characterized. Additionally, a large number of cases have shown the presence of a high number of normal cells and a diploid distribution DNA [22]. According to Petersson et al. [16] this variability and lack of recurrent abnormalities can be due to the biphasic nature of these tumors and to the different methods of culture utilized in studies. It was demonstrated that clonal chromosomal alterations in fibroadenomas were restricted to the mesenchymal cells, and epithelial cells from the same tumor were cytogenetically normal [4,5,14]. However, clonal chromosomal alterations were also found in fibroadenoma cultures enriched for epithelial cells [13,16]. Biran et al. [23] found that different tumors require different enzyme exposure times for dissociation of epithelial cells from the stromal elements. The addition of serum to a growth medium facilitates the growth of fibroblasts but may inhibit epithelial growth. Indeed, the limited use of serum in culture has been shown not to cause massive proliferation of fibroblast, but clearly to improve the epithelial cell growth [24]. In this study, normal cells were observed in all cases analyzed, with frequencies ranging from 7% to 77.7%. In most of the cases described in the literature, the frequencies of normal cells are higher than the ones observed in some of our cases. Although this can be related to the number of cells analyzed after G banding and while some of the abnormalities observed may not be representative of the in vivo situation, the biphasic nature of these tumors and the different methods of culture utilized in these studies, as mentioned, can account for this variability. It is notable, however, that the modal chromosome number was diploid for all cases analyzed in this report (data not shown). Cytogenetic studies on characterized epithelial and stromal cells of fibroadenomas are important in order to confirm and to understand the implication of the presence of karyotypically normal cells in these tumors. Clonal chromosomal alterations were observed in all cases of fibroadenomas analyzed in this study. The numerical changes involved preferentially chromosomes 22, X, 14, and 20 and the structural changes involved the chromosomes 6, 9, 11, 12, 13, 14, 20, and X. Although a high heterogeneity of chromosome alterations were observed in our study and we cannot rule out the possibility that the time in
Table 2 Karyotypes obtained in the ten cases of fibroadenomas of the breast analyzed after GTG-banding Case
Karyotype
01 02 03 04 05 06 07 08 09 10
4248, XX, 14, 21, 22 [cp10]/46,XX[1] 4446, XX, X, 5, 9, add(12)(q21q24), inv(20)(p13q11), 22[cp15]/46, XX[6] 4448, XX, X, 12, 14, mar [cp13]/46,XX[1] 47, XX, 20[2]/46,XX[7] 4147, XX, X, 6, del(6)(q23), del(6)(q23), t(12;14)(p14;q32), 17, 20, 21, mar [cp21]/46, XX [2] 4245, XX, X, del(6)(q23), i(11)(p10), 12, 17, 18, 19, 20, 21,21, 22 [cp23]/46, XX[2] 4246, XX, 17, 18, 18, 19, 22 [cp22]/46,XX [9] 4346, XX, 9, 10, 14 [cp12]/46, XX, del(9)(q23)[4]/46, XX[18] 46, XX, del(13)(q22)[4]/46, XX[11] 4246, XX, 14, 18, 20, 22 [cp15]/46, X, del(X)(q26)[4]/46, XX[21]
122
L.R. Cavalli et al. / Cancer Genetics and Cytogenetics 131 (2001) 120–124
Fig. 1. Chromosomes involved in clonal chromosomal alterations distributed per number of cases (total of 10 cases analyzed).
culture and/or the tissue culture method used may have favored the preferential growth of specific cells in vitro and, therefore, that some abnormalities observed may not be representative of the in vivo situation, some of the chromosomes involved in the clonal cytogenetic alterations were already described in the literature in other cases of fibroadenomas and benign lesions of the breast, confirming the nonrandom involvement in these benign entities. Monosomy of chromosome 22, the most frequent chromosome loss observed in our study (50% of the cases analyzed), was previously described in fibroadenomas of the breast by Tibiletti et al. [18] and in a mammary fibroepithelial hyperplasia reported by Burbano et al [25]. The monosomy of chromosome X, also observed with high frequency in this report (40% of the cases), was described in other fibroadenomas cases in the literature [18] as well as in a series of breast tumors cytogenetically analyzed [26]. Its significance, however, is unknown and it has been questioned whether they were derived from neoplastic cells [27,28]. Rearrangements on Xq26, observed in case 10, was also observed as the sole structural alteration in one fibroadenoma reported by Petersson et al. [16] as a t(X;1)(q26;p33). Gain of chromosome 20, found in case 4 of this study, was previously reported in other fibroadenomas [4,16,18] and in one case of fibroepithelial hyperplasia of the breast [25]. In case 2, a structural alteration involving chromo-
some 20, inv(20)(p13q11), was observed in 33% of the cells analyzed. This same abnormality was observed in case 4, but as a nonclonal chromosomal alteration. Calabrese et al. [9] reported a fibroadenoma with the involvement of band 20q11 as a structural alteration. Structural alterations involving chromosome 12 have been described with relative frequency in fibroadenomas [9,12,13,15] and other breast diseases [29]. Translocations involving 12p12 were observed by Calabrese et al. [9] in two fibroadenomas. In a phyllodes tumor of the breast, the region 12p12 was deleted [del(12)(p11p12)] [30], and the region 12p13 was translocated [t(3;12)(q21;p13)] [31]. Alterations at 12p12 were also observed in a fibrocystic lesion of the breast [32]. The K-RAS oncogene is localized at 12p12 and is frequently altered in mammary carcinomas [33]. Alterations involving 12q13q15, the site of the oncogenes INT1 and GLI (HGM11, 1991), have been described in subsets of pleomorphic adenomas of the salivary glands, uterine leiomyomas, and lipomas [34] and also in benign epithelial lesions [29,31], including fibroadenomas [9,12,15]. The HGMI-C gene (high mobility group protein) mapped to 12q15 [34], within the region designated MAR (multiple aberration region), was found to be rearranged in several benign mesenchymal neoplasms [35] and in the fibroadenoma described by Staats et al. [15]. In case 2 of this study, additional chromosomal material in band 12q21q24
Fig. 2. Partial karyotypes showing structural chromosomal abnormalities involving chromosomes 6, 11, 12, and 20 (arrows indicate the rearranged chromosome).
L.R. Cavalli et al. / Cancer Genetics and Cytogenetics 131 (2001) 120–124
was observed. Structural chromosomal alterations in chromosome 12 are, therefore, a recurrent and common abnormality in some of the fibroadenomas described in the literature, and further analysis on the breakpoints involved may indicate genes with relevance to fibroadenoma tumorigenesis. Translocations involving chromosomes 12 and 14 were reported by Dietrich et al. [13] in one fibroadenoma, however, the breakpoints were different from the ones observed in the present study. Translocations involving chromosome 14 were also described in fibroadenomas by Belda et al. [5] and Leuschner et al. [11] with the breakpoints affecting bands 14q24 and 14q32, respectively. Rearrangements involving the long arm of chromosome 6, were previously described in fibroadenomas [4,11,18] and in other mammary benign lesions [13,25,33,36]. Tibiletti et al. [18] observed deletions in 6q in 83.9% of the benign tumors analyzed, most of which consisted of fibroadenomas. This same rearrangement was observed with high frequency in premalignant breast lesions, as well as in mammary carcinomas by the same authors, implying that 6q alterations are among the earliest events in the pathogenesis of breast cancer. Several studies in preinvasive and invasive breast tissue have shown loss of heterozygosity at this region, indicating the presence of a tumor suppressor gene [37–39]. A series of genes associated with mammary tumor progression has been mapped at 6q, including growth inhibiting and senescence promoting genes [40]. Rearrangements involving 6q are also common in ovarian carcinomas [41], and allelotyping studies have indicated several distinct loci of allelic imbalance at this region [41,42]. In summary, according to the cytogenetic data available on fibroadenomas and other benign breast lesions in the literature, some recurrent abnormalities, such as structural alterations of 6q or chromosome 12, are starting to emerge. Characterization of these recurrent abnormalities in future studies using molecular analysis may additionally clarify their association with a particular type of benign breast pathology. Finally, although some of the abnormalities described in our study have been described in epithelial malignant breast tumors as well, and may indicate a preneoplastic condition, it is of interest that the clinical follow-up (5–10 years) for all of these patients has indicated no sign of recurrent disease to date. Additional cases of fibroadenomas as well as other biphasic tumors should be investigated in order to understand the meaning of the chromosome alterations observed. The finding of similarity between some chromosome alterations in fibroadenomas and carcinomas of the breast should be considered in an attempt to verify the progression risk (if any) and if it the same genetic pathway is involved in the etiology of both of these tumors. Acknowledgments The authors thank Dr. Bassem Haddad for the careful reading of the manuscript and the staff of the Oncology Service from the Hospital Nossa Senhora das Graças, Curitiba,
123
for providing the samples. This work was partially supported by a research grant from CNPq (National Research Council for Scientific and Technologic Development). References [1] Harris JR, Hellman S, Henderson IC, Kinne DW. Breast Diseases. 2nd Ed. Philadelphia: J.B. Lippincott; 1991. [2] Koerner FC, O’Connell JX. Fibroadenoma: morphological observations and a theory of pathogenesis. Pathol Annual 1994;29:1–19. [3] Noguchi S, Motomura K, Inaji H, Imaoka S, Koyama H. Clonal analysis of fibroadenoma and phyllodes tumor of the breast. Cancer Res 1993;53:4071–4. [4] Fletcher JA, Pinkus GS, Weidner N, Morton CC. Lineage-restricted clonality in biphasic solid tumors. Am J Pathol 1991;138:1199–207. [5] Belda F, Lester SC, Pinkus JK, Pinkus GS, Fletcher JA. Lineagerestricted chromosome translocation in a benign fibrous tumor of the breast. Hum Pathol 1993;24:923–7. [6] Noguchi S, Yokouchi H, Aihara T, Motomura K, Inaji H, Imaoka S, Koyama H. Progression of fibroadenomas to phyllodes tumor demonstrated by clonal analysis. Cancer 1995;76:1779–85. [7] McDivitt RW, Stevens JA, Lee NC, Wingo PA, Rubin GL, Gersell D and the Cancer and Steroid Hormone Study Group. Histologic types of benign breast disease and the risk for breast cancer. Cancer 1992; 69:1408–14. [8] Zhang R, Wiley J, Howard SP, Meisner LF, Gould MN. Rare clonal karyotypic variants in primary cultures of human breast carcinoma. Cancer Res 1989;49:444–9. [9] Calabrese G, Di Virgilio C, Cianchetti E, Franchi PG, Stuppia L, Parruti G, Bianchi PG, Palka G. Chromosome abnormalities in breast fibroadenomas. Genes Chromosom Cancer 1991;3:202–4. [10] Stephenson CF, Davis RI, Moore GE, Sandberg AA. Cytogenetic and fluorescence in situ hybridization analysis of breast fibroadenomas. Cancer Genet Cytogenet 1992;63:32–6. [11] Leuschner E, Meyer-Bolte K, Caselitz J, Bartnitzke S, Bullerdiek J. Fibroadenoma of the breast showing a translocation (8;14), a ring chromosome and two markers involving parts of chromosome 11. Cancer Genet Cytogenet 1994;76:145–7. [12] Ozisik YY, Meloni AM, Stephenson CF, Peier A, Moore GE, Sandberg AA. Chromosome abnormalities in breast fibroadenomas. Cancer Genet Cytogenet 1994;77:125–8. [13] Dietrich CU, Pandis N, Teixeira MR, Bardi G, Gerdes A, Andersen JA, Heim S. Chromosome abnormalities in benign hyperproliferative disorders of epithelial and stromal breast tissue. Int J Cancer 1995;60: 49–53. [14] Rohen C, Staats B, Bonk U, Bartnizke S, Bullerdiek J. Significance of clonal chromosome aberrations in breast fibroadenomas. Cancer Genet Cytogenet 1996;87:152–5. [15] Staats B, Bonk U, Wanschura S, Hanisch P, Schoenmakers EFPM, Van de Vem WJM, Bartnitzke S, Bullerdiek J. A fibroadenoma with a t(4;12)(q27;q15) affecting the HMGI-C gene, a member of the high mobility group protein gene family. Breast Cancer Res Treat 1996; 38:299–303. [16] Petersson C, Pandis N, Rizou H, Mertens F, Dietrich CU, Adeyinka A, Idvall I, Bondeson L, Georgiou G, Ingvar C, Heim S, Mitelman F. Karyotypic abnormalities in fibroadenomas of the breast. Int J Cancer 1997;70:282–6. [17] Dal Cin P, Pauwels P, Moerman P, Qi H, Van Den Berghe H. Hyperdiploidy in benign breast lesions. Cancer Genet Cytogenet 1998;101:162–3. [18] Tibiletti MG, Sessa F, Bernasconi B, Cerutti R, Broggi B, Furlan D, Acquati F, Bianchi M, Russo A, Capella C, Taramelli R. A large 6q deletion is a common cytogenetic alteration in fibroadenomas, premalignant lesions, and carcinomas of the breast. Clin Cancer Res 2000;6:1422–31. [19] Yunis JJ. New chromosome techniques in the study of human neoplasia. Hum Pathol 1981;12:540–9.
124
L.R. Cavalli et al. / Cancer Genetics and Cytogenetics 131 (2001) 120–124
[20] Scheres VMJC. Identification of two Robertsonian translocation with a Giemsa banding technique. Hum Genet 1972;15:253–6. [21] ISCN. An international system for human cytogenetic nomenclature. Mitelman F, editor. Basel: S. Karger, 1995. [22] Ried T, Just KE, Holtgreve-Grez H, duManoir S, Speicher MR, Schröck E, Latham C, Blegen H, Zetterberg A, Cremer T, Auer G. Comparative genomic hybridization of formalin-fixed, paraffinembedded breast tumors reveals different patterns of chromosome gains and losses in fibroadenomas and diploid and aneuploid carcinomas. Cancer Res 1995;55:5415–23. [23] Biran S, Vlodavsky I, Fuks Z, Lijovetzky G, Horowitz AT. Growth of human mammary carcinoma cells from biopsy specimens in serumfree medium on extracellular matrix. Int J Cancer 1986;38:345–54. [24] Pandis N, Heim S, Bardi G, Limon J, Mandahl N, Mitelman F. Improved technique for short-term culture and cytogenetic analysis of human breast cancer. Genes Chromosom Cancer 1992;5:14–20. [25] Burbano RR, Medeiros A, Amorim MIM, Lima EM, Mello A, Barbieri Neto J, Casartelli C. Cytogenetics of epithelial hyperplasias of the human breast. Cancer Genet Cytogenet 2000;119:62–6. [26] Cavalli LR, Cavaliéri LMB, Ribeiro LA, Cavalli IJ, Silveira R, Rogatto SR. Cytogenetic evaluation of 20 primary breast carcinomas. Hereditas 1997;126:261–8. [27] Johansson B, Heim S, Mandahl N, Mertens F, Mitelman F. Trisomy 7 in nonneoplastic cells. Genes Chromosom Cancer 1993;6:199–205. [28] Rogatto SR, Rainho CA, Barbieri-Neto J, Casartelli C. Loss of the sex chromosome in human neoplasias of the nervous system. Rev Bras Genética 1994;17:215–22. [29] Rohen C, Bonk U, Staats B, Bartnitzke S, Bullerdiek J. Two human breast tumors with translocations involving 12q13-15 as the sole cytogenetic abnormality. Cancer Genet Cytogenet 1993;69:68–71. [30] Dietrich CU, Pandis N, Bardi G, Teixeira MR, Soukhikh T, Petersson C, Andersen JA, Heim S. Karyotypic changes in phyllodes tumors of the breast. Cancer Genet Cytogenet 1994;78:200–6. [31] Dietrich CU, Pandis N, Rizou H, Petersson C, Bardi G, Qvist H, Apostolikas N, Boher PJ, Andersen JÁ, Idvall I, Mitelman F, Heim S. Cytogenetic findings in phyllodes tumors of the breast: karyotypic complexity differentiates between malignant and benign tumors. Hum Pathol 1997;28:1379–82.
[32] Nielsen KV, Briand P. Cytogenetics analysis of in vitro karyotype evolution in a cell line established from nonmalignant human mammary epithelium. Cancer Genet Cytogenet 1989;39:103–18. [33] Mars WM, Saunders GF. Chromosomal abnormalities in human breast cancer. Cancer Metastases Rev 1990;9:35–43. [34] Wanschura S, Kazmierczak B, Schoenmakers E, Meyen E, Bartnitzke S, Van de Vem W, Bullerdiek J, Schloot W. Regional fine mapping of the multiple aberration region involved in uterine leiomyoma, lipoma and pleomorphic adenoma of the salivary gland to 12q15. Genes Chromosom Cancer 1995;14:68–70. [35] Schoenmakers EFPM, Wanschura S, Mols R, Bullerdiek J, Van den Berghe H, Van de Ven WJM. Recurrent rearrangements in the high mobility group protein gene, HGMI-C, in benign mesenchymal tumors. Nat Genet 1995;10:436–44. [36] Birdsall SH, Maclennan KA, Gusterson BA. t(6;12)(q23;q13) and t(10;16)(q22;p11) in a phyllodes tumor of breast. Cancer Genet Cytogenet 1992;60:74–7. [37] Theile M, Seitz S, Arnold W, Jandrig B, Frege R, Schlag P, Haensch W, Guski H, Winzer K, Barrett J, Scherneck S. A defined chromosome 6q fragment (at D6S310) harbours a putative tumor supressor gene for breast cancer. Oncogene 1996;13:677–85. [38] Chappel SA, Walsh T, Walker RA, Shaw JA. Loss of heterozygosity at chromosome 6q in preinvasive and early invasive breast carcinomas. Br J Cancer 1997;75:1324–9. [39] Rodriguez C, Causse A, Ursule E, Theillet C. At least five regions of imbalance on 6q in breast tumors, combining losses and gains. Genes Chromosom Cancer 2000;27:76–84. [40] Banga S, Kim S, Hubbard K, Dasgupta T, Jha K, Patsalis P, Hauptschein R, Gamberi B, Dalla Faveera R, Kraemer P, Ozer H. SEN 6, a locus for SV40-mediated immortalization of human cells maps to 6q26-27. Oncogene 1997;14:313–21. [41] Saito S, Saito H, Koi S, Sagae S, Kudo R, Saito J, Noda K, Nakamura Y. Fine-scale deletion mapping of the distal long arm of chromosome 6 in 70 human ovarian cancers. Cancer Res 1992;52:5815–17. [42] Colitti CV, Rodabaugh KJ, Welch WR, Berkowitz RS, Mok SC. A novel 4cM minimal deletion unit on chromosome 6q25.1-q25.2 associated with high grade invasive epithelial ovarian carcinomas. Oncogene 1998;16:555–9.