Open Access
Open Peer Review

This article has Open Peer Review reports available.

How does Open Peer Review work?

Aberrant DNA methylation of cancer-related genes in giant breast fibroadenoma: a case report

  • Diego M Marzese1, 2,
  • Francisco E Gago2, 3,
  • Javier I Orozco2, 3,
  • Olga M Tello3,
  • María Roqué1 and
  • Laura M Vargas-Roig2, 4Email author
Journal of Medical Case Reports20115:516

https://doi.org/10.1186/1752-1947-5-516

Received: 17 March 2011

Accepted: 18 October 2011

Published: 18 October 2011

Abstract

Introduction

Giant fibroadenoma is an uncommon variant of benign breast lesions. Aberrant methylation of CpG islands in promoter regions is known to be involved in the silencing of genes (for example, tumor-suppressor genes) and appears to be an early event in the etiology of breast carcinogenesis. Only hypermethylation of p16INK4a has been reported in non-giant breast fibroadenoma. In this particular case, there are no previously published data on epigenetic alterations in giant fibroadenomas. Our previous results, based on the analysis of 49 cancer-related CpG islands have confirmed that the aberrant methylation is specific to malignant breast tumors and that it is completely absent in normal breast tissue and breast fibroadenomas.

Case presentation

A 13-year-old Hispanic girl was referred after she had noted a progressive development of a mass in her left breast. On physical examination, a 10 × 10 cm lump was detected and axillary lymph nodes were not enlarged. After surgical removal the lump was diagnosed as a giant fibroadenoma. Because of the high growth rate of this benign tumor, we decided to analyze the methylation status of 49 CpG islands related to cell growth control. We have identified the methylation of five cancer-related CpG islands in the giant fibroadenoma tissue: ESR1, MGMT, WT-1, BRCA2 and CD44.

Conclusion

In this case report we show for the first time the methylation analysis of a giant fibroadenoma. The detection of methylation of these five cancer-related regions indicates substantial epigenomic differences with non-giant fibroadenomas. Epigenetic alterations could explain the higher growth rate of this tumor. Our data contribute to the growing knowledge of aberrant methylation in breast diseases. In this particular case, there exist no previous data regarding the role of methylation in giant fibroadenomas, considered by definition as a benign breast lesion.

Introduction

Fibroadenoma represents the most frequent breast lesion in adolescents and young women with the giant fibroadenoma (GF) being an uncommon variant. GFs, which occur mostly in adolescent girls, are characterized by their large size (more than 5 cm). They are encapsulated masses and generally asymptomatic. Their rapid growth (between two and five months) is associated with skin congestion and ocasionally ulceration. It is thought that increased estrogen receptor sensitivity is responsible for the etiology of GF [1].

Aberrant methylation of CpG islands (CpGIs) in promoter regions is known to be involved in the silencing of tumor-suppressor genes, steroid receptors, cell adhesion molecules and cell cycle regulator genes and appears to be an early event in the etiology of breast carcinogenesis [2]. The aberrant methylation of cell cycle regulator genes leads to a higher proliferation rate [3].

Our previous results, based on the analysis of 49 cancer-related CpGIs, have confirmed that the aberrant methylation is specific to malignant breast tumors and that it is completely absent in normal breast tissue and breast fibroadenomas [4]. Other authors have reported aberrant methylation of p16INK4a not only in malignant breast lesions but also in fibroadenoma and normal mammary tissues [5]. There are no previous data of epigenetic alterations in giant fibroadenomas. The established precursors of breast carcinoma are atypical ductal hyperplasia, ductal carcinoma in situ , and lobular neoplasia. The malignant transformation of a fibroadenoma is a rare event, with about 100 cases reported in the world literature. Despite this fact we decided to analyze the methylation status of a GF which is a rapidly growing benign breast lesion [6], because the methylation of the analyzed genes is associated with a greater capacity for cell growth [3].

Case presentation

A 13-year-old Hispanic girl was referred after she had noted the progressive development of a mass in her left breast. On physical examination, a 10 × 10 cm lump was detected. Her axillary lymph nodes were not enlarged. Surgery was performed and a GF was removed. At present, with a follow-up of three years, both breasts are symmetrical, normally developed, and no signs of recurrence have been detected at clinical evaluations.

Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay was performed on the DNA obtained from the GF to study the methylation status of the 49 CpGIs (Table 1). We have previously analyzed these regions in invasive ductal carcinomas, breast fibroadenomas and normal mammary tissue [4]. The MS-MLPA Kits ME001 and ME002 were used according to the manufacturer's recommendations (MRC-Holland, Amsterdam, Netherlands) with minimal modifications [4].
Table 1

CpG Islands analyzed

 

Gene

Region

 

Gene

Region

 

Gene

Region

 

Gene

Region

 

Gene

Region

1

APC

-21 bp

11

CDH13

186 bp

21

IGSF4

-56 bp

31

p73

+258 bp

41

RASSF1

+46 bp

2

ATM

+309 bp

12

CHFR

-103 bp

22

IGSF4

-294 bp

32

p73

+25 bp

42

RB1

-226 bp

3

ATM

+138 bp

13

CHFR

-96bp

23

MGMT

-463 bp

33

PAX5

-120 bp

43

RB1

-449 bp

4

BRCA1

-20bp

14

DAPK1

+527 bp

24

MLH1

+55 bp

34

PAX6

-52 bp

44

STK11

+416 bp

5

BRCA1

+86bp

15

ESR1

+244 bp

25

MLH1

-320 bp

35

PTEN

-813 bp

45

THBS1

-791 bp

6

BRCA2

+221 bp

16

FHIT

+225 bp

26

p15

+473 bp

36

PTEN

-66 bp

46

TIMP3

+1019 bp

7

BRCA2

+138 bp

17

GATA5

+271 bp

27

p16

-817 bp

37

PYCARD

+437 bp

47

VHL

+115 bp

8

CASP8

+291bp

18

GSTP1

+148 bp

28

p16

+200 bp

38

RARβ

-357 bp

48

VHL

-3 bp

9

CD44

+411 bp

19

GSTP1

+468 bp

29

P27

+307 bp

39

RARβ

-180 bp

49

WT1

-210 bp

10

CD44

+28 bp

20

HIC1

-6 bp

30

P53

+100 bp

40

RASSF1

-136 bp

   

The table shows the 49 genomic regions tested during the study. Positive and negative signs are related to the transcription start base pair.

The immunohistochemical procedure was performed as reported previously using the monoclonal antibody clone ER88 (Biogenex, CA, USA) against estrogen receptor alpha protein [7].

We have detected aberrant methylation in five cancer-related CpGIs, that is estrogen receptor-α [ESR1 (+244bp)], O6-methylguanine-DNA methyltransferase [MGMT (-463bp)], Wilms' Tumor-1 [WT-1 (-146bp)], Breast Cancer 2 [BRCA2 (+138bp)] and Hermen Antigen [CD44 (+28bp)] (Figure 1). As a control we have analyzed six normal breast tissues and three breast fibroadenomas from 21-, 23- and 29-year-old patients. None of these samples showed methylation in any of the 49 CpGIs.
Figure 1

Detection of aberrant DNA methylation in the giant fibroadenoma. A: MS-MLPA analysis of DNA isolated from non-giant fibroadenoma. None of the analyzed regions are methylated. Only the PCR products from control probes are detected. B and C: MS-MLPA analysis of DNA isolated from the giant fibroadenoma. The methylation specific peaks are marked with an asterisk (*). Panel B shows the presence of methylation in BRCA2, CD44 and ESR1 genes and panel C shows the methylation of WT1, ESR1 and MGMT genes.

In order to evaluate the effect of the aberrant methylation on the level of protein expression in the fibroadenoma, we investigated the expression of ERα protein observing a moderate intensity in only 15% of the fibroadenoma epithelial cells (Figure 2).
Figure 2

Immunostaining of ERα protein. The figure shows the staining in the nuclei of a few epithelial cells of the giant fibroadenoma (400x).

Discussion

To the best of our knowledge, the only reported aberrant methylation in fibroadenomas is in gene p16INK4a. Our previous results analyzing a 49-gene regions panel which does not include the same reported CpGI of p16INK4a- have not revealed aberrant methylation in benign breast lesions [4, 5].

Our finding of five aberrant methylated regions in the reported GF suggests that this type of fibroadenoma presents a different etiology than other benign breast lesions, at least regarding the methylation profile.

In invasive breast tumors we have detected from two to 23 aberrantly methylated cancer-related regions, which indicates that five affected CpGIs is not a high number for a breast carcinoma (unpublished data). The surprising novelty, however, is that this finding occurs in a benign lesion.

These five aberrant methylated genes play diverse functions in the cell: DNA reparation (MGMT and BRCA2), cell cycle control (BRCA2, WT1), proliferation (WT1, ESR1) and cell adhesion (CD44). The methylation of three of them (ESR1, MGMT and WT1) has been widely reported in breast tumors [2, 4, 8]. Methylation of WT1 has not been found in normal tissue [9]. Previous studies have reported the methylation of BRCA2 in breast tumor but to the best of our knowledge, our study is the first to find methylated BRCA2 in benign breast disease [10]. Regarding gene CD44, as far as we know, its methylation status has not been reported in mammary tissue before, even though new evidence suggests its methylation in the breast cancer cell line MCF7 [11]. Methylation of the ESR1 promoter and its first exon has been observed to be correlated with loss of the expression of ERα receptor, even though some breast cancer specimens maintain its expression (ER+) [1214]. Tests based on ERα staining in fibroadenoma reveal a pronounced heterogeneity (range between 1% and 85%) showing no age correlation [15]. Our specimen expresses 15% of ERα protein, which is considered low. Even though we are not able to establish the percentage of methylated ESR1 genes in the GF, given its heterogeneity, this low protein expression is in accordance with the determined methylated gene profile. The methylation of these five regions could be responsible in part for the high growth rate present in the analyzed GF.

Conclusions

Our data contribute to the growing knowledge of aberrant methylation in breast diseases. In this particular case there were no previously published data regarding the role of methylation in GFs, considered by definition to be a benign breast lesion. These findings should be taken into account to evaluate whether it is associated with the different etiology of non-GFs and GFs. Further studies will be necessary to draw more definitive conclusions about the meaning of the methylation de-regulation in this type of disease.

Consent

Written informed consent was obtained from the patient's next-of-kin for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.

The study was approved by the Bioethics Committee of the School of Medical Sciences, National University of Cuyo, Mendoza, Argentina.

Abbreviations

BRCA2: 

Breast Cancer 2

CD44: 

Hermen Antigen

CpGIs: 

CpG islands

ERα: 

estrogen receptor α protein

ESR1: 

estrogen receptor-α

GF: 

giant fibroadenoma

MGMT: 

O6-methylguanine-DNA methyltransferase

MS-MLPA: 

Methylation-specific multiplex ligation-dependent probe amplification

p16INK4a: 

Cyclin-dependent kinase inhibitor 2A

WT-1: 

Wilms' Tumor-1

Declarations

Acknowledgements

Funding for this study was provided by SECTyP, National University of Cuyo (06-J343) and the School of Medical Sciences, National University of Cuyo, Mendoza, Argentina.

Authors’ Affiliations

(1)
Cellular and Molecular Laboratory, IHEM-CCT-CONICET
(2)
School of Medical Sciences, National University of Cuyo
(3)
Gineco-Mamario Institute
(4)
Tumor Biology Laboratory, IMBECU-CCT-CONICET

References

  1. Gobbi D, Dall'Igna P, Alaggio R, Nitti D, Cecchetto G: Giant fibroadenoma of the breast in adolescents: report of 2 cases. J Pediatr Surg. 2009, 44: e39-41. 10.1016/j.jpedsurg.2008.11.041.View ArticlePubMedGoogle Scholar
  2. Agrawal A, Murphy RF, Agrawal DK: DNA methylation in breast and colorectal cancers. Mod Pathol. 2007, 20: 711-721. 10.1038/modpathol.3800822.View ArticlePubMedGoogle Scholar
  3. Jones PA, Baylin SB: The epigenomics of cancer. Cell. 2007, 128: 683-692. 10.1016/j.cell.2007.01.029.View ArticlePubMedPubMed CentralGoogle Scholar
  4. Marzese DM, Gago FE, Vargas-Roig LM, Roque M: Simultaneous analysis of the methylation profile of 26 cancer related regions in invasive breast carcinomas by MS-MLPA and drMS-MLPA. Mol Cell Probes. 2010, 24: 271-280. 10.1016/j.mcp.2010.05.002.View ArticlePubMedGoogle Scholar
  5. Di Vinci A, Perdelli L, Banelli B, Salvi S, Casciano I, Gelvi I, Allemanni G, Margallo E, Gatteschi B, Romani M: p16(INK4a) promoter methylation and protein expression in breast fibroadenoma and carcinoma. Int J Cancer. 2005, 114: 414-421. 10.1002/ijc.20771.View ArticlePubMedGoogle Scholar
  6. Chintamani , Khandelwal R, Tandon M, Yashwant K, Kulshresthal P, Aeron T, Bhatnagar D, Bansal A, Saxena S: Carcinoma developing in a fibroadenoma in a woman with a family history of breast cancer: a case report and review of literature. Cases Journal. 2009, 2: 9348-10.1186/1757-1626-2-9348.View ArticlePubMedPubMed CentralGoogle Scholar
  7. Vargas-Roig LM, Cuello-Carrión FD, Fernández-Escobar N, Daguerre P, Leuzzi M, Ibarra J, Gago FE, Nadin SB, Ciocca DR: Prognostic value of Bcl-2 in breast cancer patients treated with neoadjuvant anthracycline based chemotherapy. Molecular Oncology. 2008, 2: 102-111. 10.1016/j.molonc.2008.01.004.View ArticlePubMedGoogle Scholar
  8. Munot K, Bell SM, Lane S, Horgan K, Hanby AM, Speirs V: Pattern of expression of genes linked to epigenetic silencing in human breast cancer. Hum Pathol. 2006, 37: 989-999. 10.1016/j.humpath.2006.04.013.View ArticlePubMedGoogle Scholar
  9. Loeb DM, Evron E, Patel CB, Sharma PM, Niranjan B, Buluwela L, Weitzman SA, Korz D, Sukumar S: Wilms' tumor suppressor gene (WT1) is expressed in primary breast tumors despite tumor-specific promoter methylation. Cancer Res. 2001, 61: 921-925.PubMedGoogle Scholar
  10. Cucer N, Taheri S, Ok E, Ozkul Y: Methylation status of CpG islands at sites -59 to +96 in exon 1 of the BRCA2 gene varies in mammary tissue among women with sporadic breast cancer. J Genet. 2008, 87: 155-158. 10.1007/s12041-008-0023-5.View ArticlePubMedGoogle Scholar
  11. Müller I, Wischnewski F, Pantel K, Schwarzenbach H: Promoter- and cell-specific epigenetic regulation of CD44, Cyclin D2, GLIPR1 and PTEN by methyl-CpG binding proteins and histone modifications. BMC Cancer. 2010, 10: 297-10.1186/1471-2407-10-297.View ArticlePubMedPubMed CentralGoogle Scholar
  12. Ottaviano YL, Issa JP, Parl FF, Smith HS, Baylin SB, Davidson NE: Methylation of the estrogen receptor gene CpG island marks loss of estrogen receptor expression in human breast cancer cells. Cancer Res. 1994, 54: 2552-2555.PubMedGoogle Scholar
  13. Lapidus RG, Ferguson AT, Ottaviano YL, Parl FF, Smith HS, Weitzman SA, Baylin SB, Issa J-PJ, Davidson NE: Methylation of estrogen and progesterone receptor gene 5' CpG islands correlates with lack of estrogen and progesterone receptor gene expression in breast tumors. Clin Cancer Res. 1996, 2: 805-810.PubMedGoogle Scholar
  14. Hori M, Iwasaki M, Yoshimi F, Asato Y, Itabashi M: Determination of estrogen receptor in primary breast cancer using two different monoclonal antibodies, and correlation with its mRNA expression. Pathol Int. 1999, 49: 191-197. 10.1046/j.1440-1827.1999.00845.x.View ArticlePubMedGoogle Scholar
  15. Shoker BS, Jarvis C, Clarke RB, Anderson E, Munro C, Davies MPA, Sibson DR, Sloane JP: Abnormal regulation of the oestrogen receptor in benign breast lesions. J Clin Pathol. 2000, 53: 778-783. 10.1136/jcp.53.10.778.View ArticlePubMedPubMed CentralGoogle Scholar

Copyright

© Marzese et al; licensee BioMed Central Ltd. 2011

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.