Breast Cancer Research and Treatment

, Volume 114, Issue 3, pp 433–440 | Cite as

Analysis of the progression of intraductal proliferative lesions in the breast by PCR-based clonal assay

Preclinical Study


Purpose To analyze the progression in patients with a morphological diagnosis of intraductal proliferative lesions by PCR-based clonal assay. Materials and methods An X-chromosome inactivation assay was applied to explore clonal relationships in human intraductal proliferative lesions of the breast. Four groups samples, including 40 cases of usual ductal hyperplasia (UDH), 40 cases of atypical ductal hyperplasia (ADH), 29 cases of flat epithelia atypia (FEA), and 40 cases of ductal carcinoma in situ (DCIS) were selected for analysis. Thirty specimens of normal breast tissue were used as a control group. Microdissection was performed to collect the tissue samples for extraction of genomic DNA from paraffin-embedded tissues. The DNA was subjected to PCR amplification of the CAG repeats in androgen receptor (AR) gene exon I with and without prior digestion of methylation-sensitive restriction enzyme HhaI. Gel electrophoresis was used to detect the clonal nature of these four groups samples. Results The clonal analysis confirmed monoclonality in all informative samples of DCIS cells. Normal tissues and the majority (97.1%) of UDH were shown to be polyclonal. Monoclonality was revealed in 20/39 (51.3%) cases of ADH. Among 26 cases of FEA, 20 were shown to be polyclonal, while six displayed monoclonal alterations which accounted for 23.1%. Conclusion These findings reinforce recent suggestions that clonal analysis with AR gene polymerase chain reaction may be used to define the genetic relationships among the human tumor and the breast intraductal proliferative lesions. Furthermore, our observations demonstrate nearly a half ADH and the smaller part of FEA have clonal alterations, which may be neoplastic lesions. This method would shed light on genetic abnormalities that play a role in early tumorigenesis of the breast, and thus might be an adjunct in predicting the probability of breast tumor occurrence and in guiding the management of these cases.


Intraductal proliferative lesions of the breast Clonal analysis Androgen receptor PCR Breast cancer 


  1. 1.
    Dupont WD, Page DL (1985) Risk factors for breast cancer in women with proliferative breast disease. N Engl J Med 312:146–151PubMedGoogle Scholar
  2. 2.
    Worsham MJ, Raju U, Lu M, Kapke A, Cheng J, Wolman SR (2007) Multiplicity of benign breast lesions is a risk factor for progression to breast cancer. Clin Cancer Res 13:5474–5479PubMedCrossRefGoogle Scholar
  3. 3.
    Hartmann LC, Sellers TA, Frost MH, Lingle WL, Degnim AC, Ghosh K, Vierkant RA, Maloney SD, Pankratz VS, Hillman DW, Suman VJ, Johnson J, Blake C, Tlsty T, Vachon CM, Melton LJ III, Visscher DW (2005) Benign breast disease and the risk of breast cancer. N Engl J Med 353:229–237PubMedCrossRefGoogle Scholar
  4. 4.
    Degnim AC, Visscher DW, Berman HK, Frost MH, Sellers TA, Vierkant RA, Maloney SD, Pankratz VS, de Groen PC, Lingle WL, Ghosh K, Penheiter L, Tlsty T, Melton LJ III, Reynolds CA, Hartmann LC (2007) Stratification of breast cancer risk in women with atypia: a Mayo cohort study. J Clin Oncol 25:2671–2677PubMedCrossRefGoogle Scholar
  5. 5.
    Tavassoli FA, Devilee P (2003) Intraductal proliferative lesions. Pathology and genetics of tumors of the breast and female genetic organs, the 1st version. International Agency for Research on Cancer (IARC) Press, pp 63–73Google Scholar
  6. 6.
    Amari M, Moriya T, Ishida T, Harada Y, Ohnuki K, Takeda M, Sasano H, Horii A, Ohuchi N (2003) Loss of heterozygosity analyses of asynchronous lesions of ductal carcinoma in situ and invasive ductal carcinoma of the human breast. Jpn J Clin Oncol 33:556–562PubMedCrossRefGoogle Scholar
  7. 7.
    Shaaban AM, Sloane JP, West CR, Moore FR, Jarvis C, Williams EM, Foster CS (2002) Histopathologic types of benign breast lesions and the risk of breast cancer: case–control study. Am J Surg Pathol 26:421–430PubMedCrossRefGoogle Scholar
  8. 8.
    Simpson PT, Gale T, Reis-Filho JS, Jones C, Parry S, Sloane JP, Hanby A, Pinder SE, Lee AH, Humphreys S, Ellis IO, Lakhani SR (2005) Columnar cell lesions of the breast: the missing link in breast cancer progression? A morphological and molecular analysis. Am J Surg Pathol 29:734–746PubMedCrossRefGoogle Scholar
  9. 9.
    Dabbs DJ, Carter G, Fudge M, Peng Y, Swalsky P, Finkelstein S (2006) Molecular alterations in columnar cell lesions of the breast. Mod Pathol 19:344–349PubMedCrossRefGoogle Scholar
  10. 10.
    Abdel-Fatah TM, Powe DG, Hodi Z, Lee AH, Reis-Filho JS, Ellis IO (2007) High frequency of coexistence of columnar cell lesions, lobular neoplasia, and low grade ductal carcinoma in situ with invasive tubular carcinoma and invasive lobular carcinoma. Am J Surg Pathol 31:417–426PubMedCrossRefGoogle Scholar
  11. 11.
    Simpson PT, Reis-Filho JS, Gale T, Lakhani SR (2005) Molecular evolution of breast cancer. J Pathol 205:248–254PubMedCrossRefGoogle Scholar
  12. 12.
    Larson PS, de las Morenas A, Cerda SR, Bennett SR, Cupples LA, Rosenberg CL (2006) Quantitative analysis of allele imbalance supports atypical ductal hyperplasia lesions as direct breast cancer precursors. J Pathol 209:307–316PubMedCrossRefGoogle Scholar
  13. 13.
    Di Cristofano C, Mrad K, Zavaglia K, Bertacca G, Aretini P, Cipollini G, Bevilacqua G, Ben Romdhane K, Cavazzana A (2005) Papillary lesions of the breast: a molecular progression? Breast Cancer Res Treat 90:71–76PubMedCrossRefGoogle Scholar
  14. 14.
    Nyante SJ, Devries S, Chen YY, Hwang ES (2004) Array-based comparative genomic hybridization of ductal carcinoma in situ and synchronous invasive lobular cancer. Hum Pathol 35:759–763PubMedCrossRefGoogle Scholar
  15. 15.
    Hwang ES, DeVries S, Chew KL, Moore DH II, Kerlikowske K, Thor A, Ljung BM, Waldman FM (2004) Patterns of chromosomal alterations in breast ductal carcinoma in situ. Clin Cancer Res 10:5160–5167PubMedCrossRefGoogle Scholar
  16. 16.
    Knudson AG Jr (1985) Hereditary cancer, oncogenes, and antioncogenes. Cancer Res 45:1437–1443PubMedGoogle Scholar
  17. 17.
    Su Q, Liu Q, Wang SF (2002) The clonality analysis based on X-chromosome gene polymorphism and its application. Zhonghua Binglixue Zazhi 31:162–164Google Scholar
  18. 18.
    Diaz-Cano SJ, Blanes A, Wolfe HJ (2001) PCR techniques for clonality assays. Diagn Mol Pathol 10:24–33PubMedCrossRefGoogle Scholar
  19. 19.
    Allen CR, Zoghbi HY, Moseley AB, Rosenblatt HM, Belmont JW (1992) Methylation of HpaII and HhaI sites near the polymorphic CAG repeat in the human androgen receptor gene correlates with X-chromosome inactivation. Am J Hum Genet 51:1229PubMedGoogle Scholar
  20. 20.
    Yakirevich E, Jackson CL, Meitner PA, MacKenzie D, Tavares R, Robinson-Bostom L, DeLellis RA, Resnick MB (2007) Analysis of T-cell clonality using laser capture microdissection and high-resolution microcapillary electrophoresis. J Mol Diagn 9:490–497PubMedCrossRefGoogle Scholar
  21. 21.
    Wu Y, Basir Z, Kajdacsy-Balla A (2003) Resolution of clonal origins for endometriotic lesions using laser capture microdissection and the human androgen receptor (HUMARA) assay. Fertil Steril 79:710–717PubMedCrossRefGoogle Scholar
  22. 22.
    Harada M, Suzuki M, Ikeda T, Kaneko T, Harada S, Fukayama M (1997) Clonality in nevo-cellular nevus and melanoma: an expression based clonality analysis at the X linked genes by polymerase chain reaction. J Invest Dermatol 109:656–660PubMedCrossRefGoogle Scholar
  23. 23.
    Cheng L, Jones TD, McCarthy RP, Eble JN, Wang M, MacLennan GT, Lopez-Beltran A, Yang XJ, Koch MO, Zhang S, Pan CX, Baldridge LA (2005) Molecular genetic evidence for a common clonal origin of urinary bladder small cell carcinoma and co-existing urothelial carcinoma. Am J Pathol 166:1533–1539PubMedGoogle Scholar
  24. 24.
    Jones TD, Eble JN, Wang M, MacLennan GT, Delahunt B, Brunelli M, Martignoni G, Lopez-Beltran A, Bonsib SM, Ulbright TM, Zhang S, Nigro K, Cheng L (2005) Molecular genetic evidence for the independent origin of multifocal papillary tumors in patients with papillary renal cell carcinomas. Clin Cancer Res 11:7226–7233PubMedCrossRefGoogle Scholar
  25. 25.
    Ochiai T, Urata Y, Yamano T, Yamagishi H, Ashihara T (2000) Clonal expansion in evolution of chronic hepatitis to hepatocellular carcinoma as seen at an X-chromosome locus. Hepatology 31:615–621PubMedCrossRefGoogle Scholar
  26. 26.
    Katona TM, Jones TD, Wang M, Abdul-Karim FW, Cummings OW, Cheng L (2006) Molecular evidence for independent origin of multifocal neuroendocrine tumors of the enteropancreatic axis. Cancer Res 66:4936–4942PubMedCrossRefGoogle Scholar
  27. 27.
    Kuijper A, Buerger H, Simon R (2002) Analysis of the progression of fibroepithelial tumors of the breast by PCR-based clonality assay. J Pathol 197:575–581PubMedCrossRefGoogle Scholar
  28. 28.
    Endoh Y, Tamura G, Kato N, Motoyama T (2001) Apocrine adenosis of the breast: clonal evidence of neoplasia. Histopathology 38:221–224PubMedCrossRefGoogle Scholar
  29. 29.
    Reis-Filho JS, Lakhani SR (2003) The diagnosis and management of pre-invasive breast disease: genetic alterations in pre-invasive lesions. Breast Cancer Res 5:313–319PubMedCrossRefGoogle Scholar
  30. 30.
    Wrensch MR, Petrakis NL, Miike R, King EB, Chew K, Neuhaus J, Lee MM, Rhys M (2001) Breast cancer risk in women with abnormal cytology in nipple aspirates of breast fluid. J Natl Cancer Inst 93:1791–1798PubMedCrossRefGoogle Scholar
  31. 31.
    Moinfar F, Man YG, Bratthauer GL, Ratschek M, Tavassoli FA (2000) Genetic abnormalities in mammary ductal intraepithelial neoplasia-flat type (‘clinging ductal carcinoma in situ’): a simulator of normal mammary epithelium. Cancer 88:2072–2081PubMedCrossRefGoogle Scholar
  32. 32.
    Fraser JL, Raza S, Chorny K, Connolly JL, Schnitt SJ (2000) Immunophenotype of columnar alteration with prominent apical snouts and secretions (CAPSS). Lab Invest 80:21AGoogle Scholar
  33. 33.
    Ghofrani M, Tapia B, Tavassoli F (2006) Discrepancies in the diagnosis of intraductal proliferative lesions of the breast and its management implications: results of a multinational survey. Virchows Arch 449:609–616PubMedCrossRefGoogle Scholar
  34. 34.
    Kunju LP, Kleer CG (2007) Significance of flat epithelial atypia on mammotome core needle biopsy: should it be excised? Hum Pathol 38:35–41PubMedCrossRefGoogle Scholar
  35. 35.
    Anderson E, Clarke RB (2004) Steroid receptors and cell cycle in normal mammary epithelium. J Mammary Gland Biol Neoplasia 9:3–13PubMedCrossRefGoogle Scholar
  36. 36.
    Gompel A, Somai S, Chaouat M, Kazem A, Kloosterboer HJ, Beusman I, Forgez P, Mimoun M, Rostene W (2000) Hormonal regulation of apoptosis in breast cells and tissues. Steroids 65:593–598PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2008

Authors and Affiliations

  • Qi Yu
    • 1
    • 2
  • Yun Niu
    • 1
  • Yong Yu
    • 1
  • XiuMin Ding
    • 1
  • YuRong Shi
    • 1
  1. 1.National Key Laboratory of Breast Cancer Research (Ministry of Education), Cancer Institute and HospitalTianjin Medical UniversityTianjinChina
  2. 2.Department of OncologyJinghai HospitalTianjinChina

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