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DNA methylation profiling of phyllodes and fibroadenoma tumours of the breast

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Abstract

Phyllodes tumours and cellular fibroadenomas are both fibroepithelial tumours of the breast. Phyllodes tumours, unlike fibroadenomas, have the ability to recur and metastasise. Although these lesions can be distinguished by their stromal cellularity, mitotic index, presence or absence of stromal overgrowth and cellular atypia, there is overlap and not infrequently a definitive diagnosis cannot be made, particularly on biopsy. We sought to evaluate whether DNA promoter methylation profiling using selected genes known to be methylated in cancer would allow us to learn more about the biology of these tumours, and whether it could identify methylation markers that could differentiate phyllodes tumours from fibroadenomas and/or distinguish phyllodes tumours of different grades. Methylation-sensitive high resolution melting (MS-HRM) was used to screen promoter DNA methylation changes in 86 phyllodes tumours (15 benign, 28 borderline, 43 malignant) and 26 fibroadenomas. A panel of 11 genes (RASSF1A, TWIST1, APC, WIF1, MGMT, MAL, RARβ, CDKN2A, CDH1, TP73 and MLH1) was tested. Methylation status was correlated with histology and with clinicopathological parameters. Five of the gene promoters showed some methylation in a proportion of phyllodes tumours; RASSF1A, 45.3%; TWIST1, 10.7%; APC, 4.1%; WIF1, 2.9% and MGMT, 1.3%. Only two genes showed any methylation in fibroadenomas usually at background levels; RASSF1A, 53.8% and MGMT, 8.3%. No CDKN2A methylation was observed in either tumour type, contrary to previous reports. Overall, the methylation patterns differed little from that which might be seen in normal cells. However, significant levels of methylation of RASSF1A (24.4%) and TWIST1 (7.1%) was observed in some phyllodes tumours. Elevated RASSF1A and/or TWIST1 methylation was significantly associated with phyllodes tumours compared with fibroadenomas (P = 0.02), TWIST1 methylation correlated with increasing malignancy in phyllodes tumours (P < 0.001). In conclusion, assessment of methylation of RASSF1A and TWIST1 may aid in the diagnosis of phyllodes tumours. The absence of frequent methylation in fibroadenomas supports a non-neoplastic origin.

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References

  1. McKenna AM, Pintilie M, Youngson B, Done SJ (2007) Quantification of the morphologic features of fibroepithelial tumors of the breast. Arch Pathol Lab Med 131(10):1568–1573

    PubMed  Google Scholar 

  2. Guerrero MA, Ballard BR, Grau AM (2003) Malignant phyllodes tumor of the breast: review of the literature and case report of stromal overgrowth. Surg Oncol 12(1):27–37

    Article  PubMed  Google Scholar 

  3. Karim RZ, Scolyer RA, Tse GM, Tan PH, Putti TC, Lee CS (2009) Pathogenic mechanisms in the initiation and progression of mammary phyllodes tumours. Pathology 41(2):105–117

    Article  CAS  PubMed  Google Scholar 

  4. Karim RZ, Gerega SK, Yang YH, Spillane A, Carmalt H, Scolyer RA, Lee CS (2009) Phyllodes tumours of the breast: a clinicopathological analysis of 65 cases from a single institution. Breast 18(3):165–170

    Article  CAS  PubMed  Google Scholar 

  5. Jacklin RK, Ridgway PF, Ziprin P, Healy V, Hadjiminas D, Darzi A (2006) Optimising preoperative diagnosis in phyllodes tumour of the breast. J Clin Pathol 59(5):454–459

    Article  CAS  PubMed  Google Scholar 

  6. Krishnamurthy S, Ashfaq R, Shin HJ, Sneige N (2000) Distinction of phyllodes tumor from fibroadenoma: a reappraisal of an old problem. Cancer 90(6):342–349

    Article  CAS  PubMed  Google Scholar 

  7. Esteller M (2008) Molecular origins of cancer: epigenetics in cancer. N Engl J Med 358(11):1148–1159

    Article  CAS  PubMed  Google Scholar 

  8. Herman JG, Baylin SB (2003) Mechanisms of disease: gene silencing in cancer in association with promoter hypermethylation. N Engl J Med 349(21):2042–2054

    Article  CAS  PubMed  Google Scholar 

  9. Levenson VV (2007) Biomarkers for early detection of breast cancer: what, when, and where? Biochim Biophys Acta 1770(6):847–856

    CAS  PubMed  Google Scholar 

  10. House MG, Guo M, Efron DT, Lillemoe KD, Cameron JL, Syphard JE, Hooker CM, Abraham SC, Montgomery EA, Herman JG, Brock MV (2003) Tumor suppressor gene hypermethylation as a predictor of gastric stromal tumor behavior. J Gastrointest Surg 7(8):1004–1014 discussion 1014

    Article  PubMed  Google Scholar 

  11. Kawaguchi K, Oda Y, Saito T, Yamamoto H, Takahira T, Kobayashi C, Tamiya S, Tateishi N, Iwamoto Y, Tsuneyoshi M (2006) DNA hypermethylation status of multiple genes in soft tissue sarcomas. Mod Pathol 19(1):106–114

    Article  CAS  PubMed  Google Scholar 

  12. Saito K, Sakurai S, Sano T, Sakamoto K, Asao T, Hosoya Y, Nakajima T, Kuwano H (2008) Aberrant methylation status of known methylation-sensitive CpG islands in gastrointestinal stromal tumors without any correlation to the state of c-kit and PDGFRA gene mutations and their malignancy. Cancer Sci 99(2):253–259

    Article  CAS  PubMed  Google Scholar 

  13. Seidel C, Bartel F, Rastetter M, Bluemke K, Wurl P, Taubert H, Dammann R (2005) Alterations of cancer-related genes in soft tissue sarcomas: hypermethylation of RASSF1A is frequently detected in leiomyosarcoma and associated with poor prognosis in sarcoma. Int J Cancer 114(3):442–447

    Article  CAS  PubMed  Google Scholar 

  14. McShane LM, Altman DG, Sauerbrei W, Taube SE, Gion M, Clark GM (2006) REporting recommendations for tumor MARKer prognostic studies (REMARK). Breast Cancer Res Treat 100(2):229–235

    Article  PubMed  Google Scholar 

  15. Kristensen LS, Mikeska T, Krypuy M, Dobrovic A (2008) Sensitive melting analysis after real time-methylation specific PCR (SMART-MSP): high-throughput and probe-free quantitative DNA methylation detection. Nucleic Acids Res 36(7):e42

    Article  PubMed  Google Scholar 

  16. Wojdacz TK, Dobrovic A (2007) Methylation-sensitive high resolution melting (MS-HRM): a new approach for sensitive and high-throughput assessment of methylation. Nucleic Acids Res 35(6):e41

    Article  PubMed  Google Scholar 

  17. Candiloro IL, Mikeska T, Hokland P, Dobrovic A (2008) Rapid analysis of heterogeneously methylated DNA using digital methylation-sensitive high resolution melting: application to the CDKN2B (p15) gene. Epigenetics Chromatin 1(1):7

    Google Scholar 

  18. Wojdacz T, Hansen L, Dobrovic A (2008) A new approach to primer design for the control of PCR bias in methylation studies. BMC Res Notes 1(1):54

    Article  PubMed  Google Scholar 

  19. Paz MF, Fraga MF, Avila S, Guo M, Pollan M, Herman JG, Esteller M (2003) A systematic profile of DNA methylation in human cancer cell lines. Cancer Res 63(5):1114–1121

    CAS  PubMed  Google Scholar 

  20. Candiloro IL, Dobrovic A (2009) Detection of MGMT promoter methylation in normal individuals is strongly associated with the T allele of the rs16906252 MGMT promoter single nucleotide polymorphism. Cancer Prev Res (Phila Pa) 2(10):862–867

    CAS  Google Scholar 

  21. Esteller M, Corn PG, Baylin SB, Herman JG (2001) A gene hypermethylation profile of human cancer. Cancer Res 61(8):3225–3229

    CAS  PubMed  Google Scholar 

  22. Feng W, Shen L, Wen S, Rosen DG, Jelinek J, Hu X, Huan S, Huang M, Liu J, Sahin AA, Hunt KK, Bast RC Jr, Shen Y, Issa JP, Yu Y (2007) Correlation between CpG methylation profiles and hormone receptor status in breast cancers. Breast Cancer Res 9(4):R57

    Article  PubMed  Google Scholar 

  23. Moribe T, Iizuka N, Miura T, Kimura N, Tamatsukuri S, Ishitsuka H, Hamamoto Y, Sakamoto K, Tamesa T, Oka M (2009) Methylation of multiple genes as molecular markers for diagnosis of a small, well-differentiated hepatocellular carcinoma. Int J Cancer 125(2):388–397

    Article  CAS  PubMed  Google Scholar 

  24. Mulero-Navarro S, Esteller M (2008) Epigenetic biomarkers for human cancer: the time is now. Crit Rev Oncol Hematol 68(1):1–11

    Article  PubMed  Google Scholar 

  25. Kim JH, Choi YD, Lee JS, Lee JH, Nam JH, Choi C, Park MH, Yoon JH (2009) Borderline and malignant phyllodes tumors display similar promoter methylation profiles. Virchows Arch 455(6):469–475

    Article  CAS  PubMed  Google Scholar 

  26. Harvey K, Tapon N (2007) The Salvador–Warts–Hippo pathway—an emerging tumour-suppressor network. Nat Rev Cancer 7(3):182–191

    Article  CAS  PubMed  Google Scholar 

  27. Mathe E (2004) RASSF1A, the new guardian of mitosis. Nat Genet 36(2):117–118

    Article  CAS  PubMed  Google Scholar 

  28. Shivakumar L, Minna J, Sakamaki T, Pestell R, White MA (2002) The RASSF1A tumor suppressor blocks cell cycle progression and inhibits cyclin D1 accumulation. Mol Cell Biol 22(12):4309–4318

    Article  CAS  PubMed  Google Scholar 

  29. Reu FJ, Leaman DW, Maitra RR, Bae SI, Cherkassky L, Fox MW, Rempinski DR, Beaulieu N, MacLeod AR, Borden EC (2006) Expression of RASSF1A, an epigenetically silenced tumor suppressor, overcomes resistance to apoptosis induction by interferons. Cancer Res 66(5):2785–2793

    Article  CAS  PubMed  Google Scholar 

  30. Oh HJ, Lee KK, Song SJ, Jin MS, Song MS, Lee JH, Im CR, Lee JO, Yonehara S, Lim DS (2006) Role of the tumor suppressor RASSF1A in Mst1-mediated apoptosis. Cancer Res 66(5):2562–2569

    Article  CAS  PubMed  Google Scholar 

  31. Yan PS, Shi H, Rahmatpanah F, Hsiau TH, Hsiau AH, Leu YW, Liu JC, Huang TH (2003) Differential distribution of DNA methylation within the RASSF1A CpG island in breast cancer. Cancer Res 63(19):6178–6186

    CAS  PubMed  Google Scholar 

  32. Issa JP, Ottaviano YL, Celano P, Hamilton SR, Davidson NE, Baylin SB (1994) Methylation of the oestrogen receptor CpG island links ageing and neoplasia in human colon. Nat Genet 7(4):536–540

    Article  CAS  PubMed  Google Scholar 

  33. Jones PA, Laird PW (1999) Cancer epigenetics comes of age. Nat Genet 21(2):163–167

    Article  CAS  PubMed  Google Scholar 

  34. Peters I, Vaske B, Albrecht K, Kuczyk MA, Jonas U, Serth J (2007) Adiposity and age are statistically related to enhanced RASSF1A tumor suppressor gene promoter methylation in normal autopsy kidney tissue. Cancer Epidemiol Biomarkers Prev 16(12):2526–2532

    Article  CAS  PubMed  Google Scholar 

  35. Lee MS, Lowe GN, Strong DD, Wergedal JE, Glackin CA (1999) TWIST, a basic helix-loop-helix transcription factor, can regulate the human osteogenic lineage. J Cell Biochem 75(4):566–577

    Article  CAS  PubMed  Google Scholar 

  36. Maestro R, Dei Tos AP, Hamamori Y, Krasnokutsky S, Sartorelli V, Kedes L, Doglioni C, Beach DH, Hannon GJ (1999) Twist is a potential oncogene that inhibits apoptosis. Genes Dev 13(17):2207–2217

    Article  CAS  PubMed  Google Scholar 

  37. Gort EH, Suijkerbuijk KP, Roothaan SM, Raman V, Vooijs M, van der Wall E, van Diest PJ (2008) Methylation of the TWIST1 promoter, TWIST1 mRNA levels, and immunohistochemical expression of TWIST1 in breast cancer. Cancer Epidemiol Biomarkers Prev 17(12):3325–3330

    Article  CAS  PubMed  Google Scholar 

  38. Mangi AA, Smith BL, Gadd MA, Tanabe KK, Ott MJ, Souba WW (1999) Surgical management of phyllodes tumors. Arch Surg 134(5):487–492 discussion 492–483

    Article  CAS  PubMed  Google Scholar 

  39. Noguchi S, Yokouchi H, Aihara T, Motomura K, Inaji H, Imaoka S, Koyama H (1995) Progression of fibroadenoma to phyllodes tumor demonstrated by clonal analysis. Cancer 76(10):1779–1785

    Article  CAS  PubMed  Google Scholar 

  40. Klarmann GJ, Decker A, Farrar WL (2008) Epigenetic gene silencing in the Wnt pathway in breast cancer. Epigenetics 3(2):59–63

    Article  PubMed  Google Scholar 

  41. Brown AM (2001) Wnt signaling in breast cancer: have we come full circle? Breast Cancer Res 3(6):351–355

    Article  CAS  PubMed  Google Scholar 

  42. Sawyer EJ, Hanby AM, Rowan AJ, Gillett CE, Thomas RE, Poulsom R, Lakhani SR, Ellis IO, Ellis P, Tomlinson IP (2002) The Wnt pathway, epithelial-stromal interactions, and malignant progression in phyllodes tumours. J Pathol 196(4):437–444

    Article  CAS  PubMed  Google Scholar 

  43. Suzuki H, Toyota M, Carraway H, Gabrielson E, Ohmura T, Fujikane T, Nishikawa N, Sogabe Y, Nojima M, Sonoda T, Mori M, Hirata K, Imai K, Shinomura Y, Baylin SB, Tokino T (2008) Frequent epigenetic inactivation of Wnt antagonist genes in breast cancer. Br J Cancer 98(6):1147–1156

    Article  CAS  PubMed  Google Scholar 

  44. Ai L, Tao Q, Zhong S, Fields CR, Kim WJ, Lee MW, Cui Y, Brown KD, Robertson KD (2006) Inactivation of Wnt inhibitory factor-1 (WIF1) expression by epigenetic silencing is a common event in breast cancer. Carcinogenesis 27(7):1341–1348

    Article  CAS  PubMed  Google Scholar 

  45. Taniguchi H, Yamamoto H, Hirata T, Miyamoto N, Oki M, Nosho K, Adachi Y, Endo T, Imai K, Shinomura Y (2005) Frequent epigenetic inactivation of Wnt inhibitory factor-1 in human gastrointestinal cancers. Oncogene 24(53):7946–7952

    Article  CAS  PubMed  Google Scholar 

  46. Mazieres J, He B, You L, Xu Z, Lee AY, Mikami I, Reguart N, Rosell R, McCormick F, Jablons DM (2004) Wnt inhibitory factor-1 is silenced by promoter hypermethylation in human lung cancer. Cancer Res 64(14):4717–4720

    Article  CAS  PubMed  Google Scholar 

  47. Kansara M, Tsang M, Kodjabachian L, Sims NA, Trivett MK, Ehrich M, Dobrovic A, Slavin J, Choong PF, Simmons PJ, Dawid IB, Thomas DM (2009) Wnt inhibitory factor 1 is epigenetically silenced in human osteosarcoma, and targeted disruption accelerates osteosarcomagenesis in mice. J Clin Invest 119(4):837–851

    Article  CAS  PubMed  Google Scholar 

  48. Esteller M, Hamilton SR, Burger PC, Baylin SB, Herman JG (1999) Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is a common event in primary human neoplasia. Cancer Res 59(4):793–797

    CAS  PubMed  Google Scholar 

  49. Jones AM, Mitter R, Springall R, Graham T, Winter E, Gillett C, Hanby AM, Tomlinson IP, Sawyer EJ (2008) A comprehensive genetic profile of phyllodes tumours of the breast detects important mutations, intra-tumoral genetic heterogeneity and new genetic changes on recurrence. J Pathol 214(5):533–544

    Article  CAS  PubMed  Google Scholar 

  50. Di Vinci A, Perdelli L, Banelli B, Salvi S, Casciano I, Gelvi I, Allemanni G, Margallo E, Gatteschi B, Romani M (2005) p16(INK4a) promoter methylation and protein expression in breast fibroadenoma and carcinoma. Int J Cancer 114(3):414–421

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We wish to thank Elena Takano for her help on sectioning. We would also like to thank Dr Thomas Mikeska for help with designing RASSF1A MS-HRM primers and discussion. We would also like to thank the other members of the Molecular Pathology Research and Development group at the Peter MacCallum Cancer Centre for their help and support. This study was funded by a grant from the Victorian Breast Cancer Research Consortium, Australia. RZK is partially funded by the Cancer Institute of NSW (Clinical Research Fellow). MY is funded by the Victorian Cancer Biobank.

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Authors and Affiliations

  1. Molecular Pathology Research and Development Laboratory, Department of Pathology, Peter MacCallum Cancer Centre, Melbourne, VIC, 3002, Australia

    Katie T. Huang & Alexander Dobrovic

  2. Departmant of Pathology, University of Melbourne, Parkville, VIC, 3010, Australia

    Katie T. Huang, Alexander Dobrovic & Stephen B. Fox

  3. Department of Pathology, Peter MacCallum Cancer Centre, Melbourne, VIC, 3002, Australia

    Max Yan & Stephen B. Fox

  4. Department of Anatomical Pathology, Royal Prince Alfred Hospital, Camperdown, NSW, 2050, Australia

    Rooshdiya Z. Karim & C. Soon Lee

  5. Molecular and Cellular Pathology, School of Medicine, University of Queensland Centre for Clinical Research, University of Queensland, Brisbane, QLD, 4029, Australia

    Sunil R. Lakhani

  6. Pathology Queensland, The Royal Brisbane & Women’s Hospital, Brisbane, QLD, 4029, Australia

    Sunil R. Lakhani

  7. Discipline of Pathology, School of Medicine, University of Western Sydney, Camperdown, NSW, 2050, Australia

    C. Soon Lee

  8. Bosch Institute, University of Sydney, Camperdown, NSW, 2050, Australia

    C. Soon Lee

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  1. Katie T. Huang
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  2. Alexander Dobrovic
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Correspondence to Stephen B. Fox.

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Huang, K.T., Dobrovic, A., Yan, M. et al. DNA methylation profiling of phyllodes and fibroadenoma tumours of the breast. Breast Cancer Res Treat 124, 555–565 (2010). https://doi.org/10.1007/s10549-010-0970-4

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  • DOI: https://doi.org/10.1007/s10549-010-0970-4

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