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Quantitative hypermethylation of a small panel of genes augments the diagnostic accuracy in fine-needle aspirate washings of breast lesions

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Abstract

Purpose

We hypothesized that comprehensive breast cancer methylation profiling might provide biomarkers for diagnostic assessment of suspicious breast lesions using fine needle aspiration biopsy (FNA).

Experimental design

Twenty-three gene promoters were surveyed by quantitative methylation-specific PCR in bisulfite-modified DNA from 66 breast carcinomas (BCa), 31 fibroadenomas (FB) and 12 normal breast (NT) samples to define a set of genes differentially methylated in malignant and non-malignant tissues. This set was tested in 78 FNA washings obtained pre-operatively (66 malignant, 12 benign), with histopathological diagnosis. Receiver operator characteristic (ROC) curve analysis identified a gene panel which might distinguish cancer from non-cancerous lesions. Finally, this panel was validated in an independent series of FNA washings (45 cases) in which cytomorphology did not reach definitive diagnosis.

Results

In tissue samples, 14-3-3-σ, DAPK, CCND2, RASSF1A, CALCA, APC, HIN1, RARβ2, TIG1, and GSTP1 methylation levels differed significantly among BCa, FB, and NT. ROC curve analysis identified a panel of four gene loci (CCND2, RASSF1A, APC, and HIN1) that discriminated BCa from benign lesions in a set of 78 FNA washings from histologically characterized breast lesions. When this panel was tested in the validation dataset of 45 FNA washings, breast cancer was identified with perfect specificity (100%) when 3 of 4 gene loci tested positive, providing estimated added information of 91% over cytomorphologic evaluation alone.

Conclusions

Our data provide evidence that multigene methylation analysis augments diagnostic accuracy of cytological assessment of suspicious breast lesions, and might be a valuable ancillary tool for breast cancer diagnosis.

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References

  1. Sidransky D (2002) Emerging molecular markers of cancer. Nat Rev Cancer 2:210–219

    Article  PubMed  CAS  Google Scholar 

  2. Jones PA, Baylin SB (2002) The fundamental role of epigenetic events in cancer. Nat Rev Genet 3:415–428

    Article  PubMed  CAS  Google Scholar 

  3. Rountree MR, Bachman KE, Herman JG et al (2001) DNA methylation, chromatin inheritance, and cancer. Oncogene 20:3156–3165

    Article  PubMed  CAS  Google Scholar 

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

    PubMed  CAS  Google Scholar 

  5. Graff JR, Herman JG, Lapidus RG et al (1995) E-Cadherin expression is silenced by DNA hypermethylation in human breast and prostate carcinomas. Cancer Res 55:5195–5199

    PubMed  CAS  Google Scholar 

  6. Nass SJ, Herman JG, Gabrielson E et al (2000) Aberrant methylation of the estrogen receptor and E-cadherin 5’ CpG islands increases with malignant progression in human breast cancer. Cancer Res 60:4346–4348

    PubMed  CAS  Google Scholar 

  7. Esteller M, Silva JM, Dominguez G et al (2000) Promoter hypermethylation and BRCA1 inactivation in sporadic breast and ovarian tumors. J Natl Cancer Inst 92:564–569

    Article  PubMed  CAS  Google Scholar 

  8. Widschwendter M, Berger J, Hermann M et al (2000) Methylation and silencing of the retinoic acid receptor-beta2 gene in breast cancer. J Natl Cancer Inst 92:826–832

    Article  PubMed  CAS  Google Scholar 

  9. Ferguson AT, Evron E, Umbricht CB et al (2000) High frequency of hypermethylation at the 14-3-3 sigma locus leads to gene silencing in breast cancer. Proc Natl Acad Sci U S A 97:6049–6054

    Article  PubMed  CAS  Google Scholar 

  10. Umbricht CB, Evron E, Gabrielson E et al (2001) Hypermethylation of 14-3-3 sigma (stratifin) is an early event in breast cancer. Oncogene 20:3348–3353

    Article  PubMed  CAS  Google Scholar 

  11. Evron E, Umbricht CB, Korz D et al (2001) Loss of cyclin D2 expression in the majority of breast cancers is associated with promoter hypermethylation. Cancer Res 61:2782–2787

    PubMed  CAS  Google Scholar 

  12. Dammann R, Yang G, Pfeifer GP (2001) Hypermethylation of the CpG island of Ras association domain family 1A (RASSF1A), a putative tumor suppressor gene from the 3p21.3 locus, occurs in a large percentage of human breast cancers. Cancer Res 61:3105–3109

    PubMed  CAS  Google Scholar 

  13. Fackler MJ, McVeigh M, Evron E et al (2003) DNA methylation of RASSF1A, HIN1, RAR-beta, Cyclin D2 and Twist in in situ and invasive lobular breast carcinoma. Int J Cancer 107:970–975

    Article  PubMed  CAS  Google Scholar 

  14. Jerónimo C, Costa I, Martins MC et al (2003) Detection of gene promoter hypermethylation in fine needle washings from breast lesions. Clin Cancer Res 9:3413–3417

    PubMed  Google Scholar 

  15. Mehrotra J, Vali M, McVeigh M et al (2004) Very high frequency of hypermethylated genes in breast cancer metastasis to the bone, brain, and lung. Clin Cancer Res 10:3104–3109

    Article  PubMed  CAS  Google Scholar 

  16. Lehmann U, Langer F, Feist H et al (2002) Quantitative assessment of promoter hypermethylation during breast cancer development. Am J Pathol 160:605–612

    PubMed  CAS  Google Scholar 

  17. Globocan available from URL: http://www-dep.iarc.fr/

  18. Jemal A, Siegel R, Ward E et al (2006) Cancer statistics. CA Cancer J Clin 56:106–130

    Article  PubMed  Google Scholar 

  19. Hamill J, Campbell ID, Mayall F et al (2002) Improved breast cytology results with near patient FNA diagnosis. Acta Cytol 46(1):19–24

    PubMed  Google Scholar 

  20. Evron E, Dooley WC, Umbricht CB et al (2001) Detection of breast cancer cells in ductal lavage fluid by methylation-specific PCR. Lancet 357(9265):1335–1336

    Article  PubMed  CAS  Google Scholar 

  21. Pu RT, Laitala LE, Alli PM et al (2003) Methylation profiling of benign and malignant breast lesions and its application to cytopathology. Mod Pathol 16(11):1095–1101

    Article  PubMed  Google Scholar 

  22. Fackler MJ, McVeigh M, Mehrotra J et al (2004) Quantitative multiplex methylation-specific PCR assay for the detection of promoter hypermethylation in multiple genes in breast cancer. Cancer Res 64(13):4442–4452

    Article  PubMed  CAS  Google Scholar 

  23. Fackler MJ, Malone K, Zhang Z et al (2006) Quantitative multiplex methylation-specific PCR analysis doubles detection of tumor cells in breast ductal fluid. Clin Cancer Res 12(11 Pt 1):3306–3310

    Article  PubMed  CAS  Google Scholar 

  24. Page DL, Anderson TJ, Sakamoto G (1987) Infiltrating carcinoma: major histological types. In: Page DL, Anderson TJ (eds) Diagnostic histopathology of the breast. Churchill-Livingstone, Edinburgh, p 193

    Google Scholar 

  25. Elston CW, Ellis IO (1998) Assessment of histological grade. In: Elston CW, Ellis IO (eds) The breast. Churchill-Livingstone, Edinburgh, p 365

    Google Scholar 

  26. Greene FL, Page DL, Fleming ID et al (2002) Breast. In: Greene FL, Page DL, Fleming ID et al (eds) AJCC cancer staging manual, 6th edn. Springer, New York, p 221

    Google Scholar 

  27. Cairns P, Esteller M, Herman JG et al (2001) Molecular detection of prostate cancer in urine by GSTP1 hypermethylation. Clin Cancer Res 7(9):2727–2730

    PubMed  CAS  Google Scholar 

  28. Olek A, Oswald J, Walter JA (1996) A modified and improved method of bisulfite based cytosine methylation analysis. Nucleic Acids Res 24:5064–5066

    Article  PubMed  CAS  Google Scholar 

  29. Jerónimo C, Henrique R, Hoque MO et al (2004) Quantitative RARβ2 hypermethylation: a promising prostate cancer marker. Clin Cancer Res 10:4010–4014

    Article  PubMed  Google Scholar 

  30. Eads CA, Lord RV, Kurumboor SK et al (2000) Fields of aberrant CpG island hypermethylation in Barrett’s esophagus and associated adenocarcinoma. Cancer Res 60:5021–5026

    PubMed  CAS  Google Scholar 

  31. Jerónimo C, Usadel H, Henrique R et al (2001) Quantitation of GSTP1 hypermethylation distinguishes between non-neoplastic prostatic tissue and organ confined prostate adenocarcinoma. J Natl Cancer Inst 93:1747–1752

    Article  PubMed  Google Scholar 

  32. Jeronimo C, Henrique R, Hoque MO et al (2004) A quantitative promoter methylation profile of prostate cancer. Clin Cancer Res 10:8472–8478

    Article  PubMed  CAS  Google Scholar 

  33. Tokumaru Y, Nomoto S, Jerónimo C et al (2003) Biallelic inactivation of the RIZ1 gene in human gastric cancer. Oncogene 22:6954–6958

    Article  PubMed  CAS  Google Scholar 

  34. Henrique R, Jerónimo C, Hoque MO et al (2005) Frequent 14-3-3σ promoter methylation in benign and malignant prostate lesions. DNA Cell Biol 24:264–269

    Article  PubMed  CAS  Google Scholar 

  35. Henrique R, Jeronimo C, Hoque MO et al (2005) MT1G hypermethylation is associated with higher tumor stage in prostate cancer. Cancer Epidemiol Biomarkers Prev 14(5):1274–1278

    Article  PubMed  CAS  Google Scholar 

  36. Rosenbaum E, Hoque MO, Cohen Y et al (2005) Promoter hypermethylation as an independent prognostic factor for relapse in patients with prostate cancer following radical prostatectomy. Clin Cancer Res 11(23):8321–8325

    Article  PubMed  CAS  Google Scholar 

  37. Herman JG, Baylin SB (2003) Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med 349:2042–2054

    Article  PubMed  CAS  Google Scholar 

  38. Lehmann U, Celikkaya G, Hasemeier B et al (2002) Promoter hypermethylation of the death-associated protein kinase gene in breast cancer is associated with the invasive lobular subtype. Cancer Res 62:6634–6638

    PubMed  CAS  Google Scholar 

  39. Lewis CM, Cler LR, Bu DW et al (2005) Promoter hypermethylation in benign breast epithelium in relation to predicted breast cancer risk. Clin Cancer Res 11:166–172

    PubMed  CAS  Google Scholar 

  40. Eads CA, Danenberg KD, Kawakami K et al (2000) MethyLight: a high-throughput assay to measure DNA methylation. Nucleic Acids Res 28(8):E32

    Article  PubMed  CAS  Google Scholar 

  41. Issa JP, Ottaviano YL, Celano P et al (1994) Methylation of the oestrogen receptor CpG island links ageing and neoplasia in human colon. Nat Genet 7:536–540

    Article  PubMed  CAS  Google Scholar 

  42. Waki T, Tamura G, Sato M et al (2003) Age-related methylation of tumor suppressor and tumour-related genes: an analysis of autopsy samples. Oncogene 22:4128–4133

    Article  PubMed  CAS  Google Scholar 

  43. Jin Z, Tamura G, Tsuchiya T et al (2001) Adenomatous polyposis coli (APC) gene promoter hypermethylation in primary breast cancers. Br J Cancer 85:69–73

    Article  PubMed  CAS  Google Scholar 

  44. Hoque MO, Feng Q, Toure P et al (2006) Detection of aberrant methylation of four genes in plasma DNA for the detection of breast cancer. J Clin Oncol 24(26):4262–4269

    Article  PubMed  CAS  Google Scholar 

  45. Muller HM, Widschwendter A, Fiegl H et al (2003) DNA methylation in serum of breast cancer patients: an independent prognostic marker. Cancer Res 63:7641–7645

    PubMed  Google Scholar 

Download references

Acknowledgements

VLC is supported by a grant from Fundação para a Ciência e a Tecnologia (SFRH/BD/23374/2005). CJ and MRT are supported by grants from Fundação para a Ciência e a Tecnologia, [Projecto de Investigação Plurianual do Centro de Investigação do IPO-Porto]. CJ, RH and MRT are the recipients of research grants from Liga Portuguesa Contra o Cancro—Núcleo Regional do Norte, Portugal.

Under a licensing agreement between Oncomethylome Sciences, SA and the Johns Hopkins University, Dr. Sidransky is entitled to a share of royalty received by the University on sale of products described in this article. Dr. Sidransky owns Oncomethylome Sciences, SA stock, which is subject to certain restrictions under University policy. Dr. Sidransky is a paid consultant to Oncomethylome Sciences, SA and is a paid member of the company’s Scientific Advisory Board. The term of this arrangement is being managed by the Johns Hopkins University in accordance with its conflict of interest policies.

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Authors and Affiliations

  1. Department of Genetics, Portuguese Oncology Institute, Rua Dr. Antonio Bernardino Almeida, Porto, Portugal

    Carmen Jeronimo, Vera L. Costa, Luísa Filipe, Irene Pais & Manuel R. Teixeira

  2. Department of Pathology, Portuguese Oncology Institute, Porto, Portugal

    Paula Monteiro, Rui Henrique, Isabel Costa, Irene Pais & Conceição Leal

  3. Department of Gastroenterology, Portuguese Oncology Institute, Porto, Portugal

    Mário Dinis-Ribeiro

  4. Fernando Pessoa University School of Health Sciences, Porto, Portugal

    Carmen Jeronimo

  5. Department of Pathology and Molecular Immunology, Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Porto, Portugal

    Carmen Jeronimo, Rui Henrique & Manuel R. Teixeira

  6. Department of Biostatistics and Medical Informatics, Faculty of Medicine, University of Porto, Porto, Portugal

    Mário Dinis-Ribeiro

  7. Department of Otolaryngology-Head and Neck Surgery, Head and Neck Cancer Research Division, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    André L. Carvalho, Mohammad O. Hoque & David Sidransky

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  1. Carmen Jeronimo
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  2. Paula Monteiro
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  3. Rui Henrique
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  4. Mário Dinis-Ribeiro
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  5. Isabel Costa
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  6. Vera L. Costa
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  7. Luísa Filipe
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  8. André L. Carvalho
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  9. Mohammad O. Hoque
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  10. Irene Pais
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  11. Conceição Leal
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  12. Manuel R. Teixeira
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  13. David Sidransky
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Correspondence to Carmen Jeronimo.

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Jeronimo, C., Monteiro, P., Henrique, R. et al. Quantitative hypermethylation of a small panel of genes augments the diagnostic accuracy in fine-needle aspirate washings of breast lesions. Breast Cancer Res Treat 109, 27–34 (2008). https://doi.org/10.1007/s10549-007-9620-x

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  • DOI: https://doi.org/10.1007/s10549-007-9620-x

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