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Methylation of BRCA1 Promoter Region Is Associated with Unfavorable Prognosis in Women with Early-Stage Breast Cancer

  • Nicholas C. Hsu,

    Affiliation Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan

  • Ya-Fang Huang,

    Affiliation Cancer Center, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan

  • Kazunari K. Yokoyama,

    Affiliation Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan

  • Pei-Yi Chu,

    Affiliation Department of Pathology, St. Martin De Porres Hospital, Chiayi, Taiwan

  • Fang-Ming Chen ,

    fchen@kmu.edu.tw (FMC); mifeho@kmu.edu.tw (MFH)

    Affiliation Division of General and Gastroenterological Surgery, Department of Surgery, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan

  • Ming-Feng Hou

    fchen@kmu.edu.tw (FMC); mifeho@kmu.edu.tw (MFH)

    Affiliations Cancer Center, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan, Division of General and Gastroenterological Surgery, Department of Surgery, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan, National Sun Yat-Sen University-Kaohsiung Medical University Joint Research Center, Kaohsiung, Taiwan, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung, Taiwan

Methylation of BRCA1 Promoter Region Is Associated with Unfavorable Prognosis in Women with Early-Stage Breast Cancer

  • Nicholas C. Hsu, 
  • Ya-Fang Huang, 
  • Kazunari K. Yokoyama, 
  • Pei-Yi Chu, 
  • Fang-Ming Chen, 
  • Ming-Feng Hou
PLOS
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Correction

25 Mar 2014: The PLOS ONE Staff (2014) Correction: Methylation of BRCA1 promoter region is associated with unfavorable prognosis in women with early-stage breast cancer. PLOS ONE 9(3): e93778. https://doi.org/10.1371/journal.pone.0093778 View correction

Abstract

BRCA1-associated breast cancers are associated with particular features such as early onset, poor histological differentiation, and hormone receptor negativity. Previous studies conducted in Taiwanese population showed that the mutation of BRCA1 gene does not play a significant role in the occurrence of breast cancer. The present study explored methylation of BRCA1 promoter and its relationship to clinical features and outcome in Taiwanese breast cancer patients. Tumor specimens from a cohort of 139 early-stage breast cancer patients were obtained during surgery before adjuvant treatment for DNA extraction. Methylation of BRCA1 promoter region was determined by methylation-specific PCR and the results were related to clinical features and outcome of patients using statistical analysis. Methylation of the BRCA1 promoter was detected in 78 (56%) of the 139 tumors. Chi-square analysis indicated that BRCA1 promoter methylation correlated significantly with triple-negative (ER-/PR-/HER2-) status of breast cancer patients (p = 0.041). The Kaplan-Meier method showed that BRCA1 promoter methylation was significantly associated with poor overall survival (p = 0.026) and disease-free survival (p = 0.001). Multivariate analysis which incorporated variables of patients' age, tumor size, grade, and lymph node metastasis revealed that BRCA1 promoter methylation was associated with overall survival (p = 0.27; hazard ratio, 16.38) and disease-free survival (p = 0.003; hazard ratio, 12.19). Our findings underscore the clinical relevance of the methylation of BRCA1 promoter in Taiwanese patients with early-stage breast cancer.

Introduction

Breast cancer is the most common type of cancer and ranked fourth in cancer mortality in Taiwan [1]. Incidence of breast cancer in Taiwan has been increasing steadily and the rise can be largely attributed to the rise of newly diagnosed young patients [2]. It has been speculated that the early onset of breast cancer in Taiwanese women may be explained by the combination of Westernized diet, sedentary lifestyle, delay childbearing, and exposure to environmental chemicals.

BRCA1 is a major cancer predisposition gene that has been the subject of intense investigation since it was identified and cloned in 1994 [3]. Breast tumors arising from BRCA1 mutation acquired distinct pathological and gene expression profiles [4]. The spectrum of BRCA1 gene mutations in breast cancer patients in various populations has been investigated [5][10]. Studies conducted in Taiwan had suggested that the mutation of BRCA1 contributes little to the occurrence of breast cancer [11], [12].

Despite germ-line mutations in BRCA1 can account for a significant fraction of familial breast cancer cases, a large proportion of familial breast cancer are not related to mutations in BRCA1 or BRCA2 [13][16]. Hypermethylation of cytosine residues in CpG islands within the promoter of many tumor suppressor genes is strongly correlated with the absence of gene expression. The epigenetic transcriptional silencing provides an alternative mechanism for the loss of function of tumor suppressor gene during cancer development [17], [18]. Hypermethylation of the BRCA1 promoter has been reported in breast cancer with links to down-regulated mRNA and protein level in tumors and cell lines [19][23]. Aberrant BRCA1 promoter methylation has also been found to be associated with particular biological and clinicopathological features [24][27]. However these reports did not result in similar conclusion as the limited population examined and the high heterogeneity among breast cancer patients in these studies might lead to the inconclusive finding. The aim of the current study was to examine the prevalence and clinical relevance of BRCA1 promoter methylation in Taiwanese women with breast cancer. Our results indicated that promoter region of BRCA1 gene is frequently methylated in Taiwanese patients with early-stage breast cancer. BRCA1-methylated tumor exhibited poorer survival outcome.

Patients and Methods

Ethics Statement

The study was approved by the institutional review board of Kaohsiung Medical University Hospital, Taiwan. Written informed consent was obtained from all participants.

Study subjects

Surgically resected specimens from a cohort of one hundred thirty-nine patients with early-stage breast cancer (stages I and IIA) were collected at Kaohsiung Medical University Hospital between October 2007 and July 2009. Tissues were frozen at −80°C until the extraction of DNA. Genomic DNA was extracted using the QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions.

Methylation-specific PCR

One µg of genomic DNA was modified using a CpGenomet DNA modification kit (Chemicon, Temecula, CA) according to the manufacturer's protocol and resuspended in TE buffer. Modified DNA was amplified in a total volume of 10 µL solution containing 1x PCR buffer, 1.5 mM MgCl2, 200 ng of each primer, 0.2 mM of each dNTP and 1 U Platinum Taq Polymerase (Life Technologies, Carlsbad, CA). Primers and conditions used for the methylation-specific PCR were first developed by Esteller et al [19]. Primer sequences for unmethylated PCR were 5′- TTG GTT TTT GTG GTA ATG GAA AAG TGT-3' and 5'- CAA AAA ATC TCA ACA AAC TCA CAC CA-3' and primers for methylated reaction were 5'- TCG TGG TAA CGG AAA AGC GC-3' and 5'- AAA TCT CAA CGA ACT CAC GCC G-3'. The sense primers of the unmethylated PCR and methylated PCR begin at nucleotide position 1536 and 1543 bp of BRCA1 (GenBank sequence U37574), respectively. This region crosses the major transcription start site of BRCA1 gene [28].The amplified product of unmethylated PCR is 86 bp and that of methylated product is 75 bp.

For methylated genes, SssI treated DNA (MDA-MB-231 cells) was used as a positive control and DNA from normal lymphocytes was used as a negative control. After amplification, PCR products were then loaded and electrophoresed on 2% agarose gels, stained with ethidium bromide and visualized under UV illumination. The presence of a product in the methylated reaction indicated the presence of methylated BRCA1 genes. Tumors that were positive for both methylated and unmethylated reactions were classified as having methylated BRCA1 genes.

Immunohistochemical staining

Immunohistochemical staining was performed on representative samples. The selected paraffin-embedded sections were cut and mounted on poly-l-lysine-coated slides. Staining was carried out with a mouse monoclonal BRCA1 antibody (Clone MS110, Biocare Medical, Concord, CA) at 1∶50 dilution in a Leica Bond-max automated immunostainer (Leica Microsystems, Newcastle, UK), according to manufacturer's protocol.

Statistical analysis

Association between BRCA1 methylation status and clinicopathological characteristics was analyzed by use of Pearson chi-square test. Survival rate and disease recurrence were calculated using Kaplan-Meier analysis and compared by the Cochran-Mantel-Haenszel test. Overall survival (OS) was calculated from the time of initial diagnosis to death of any cause. Disease-free survival (DFS) was defined as the time between initial diagnosis and diseases recurrence. Patients alive or disease free at the end of the follow-up period were censored. Data from medical charts were retrieved, and the patients' outcomes were followed until January, 2012 or date of death, whichever occurred first. Potential confounding factors were adjusted and analyzed by use of the Cox proportional hazards regression model. The variables in the model included age, tumor size, lymph node metastasis, and histological grade. All statistical calculations were done using SPSS version 17.0 for windows (SPSS, Inc., Chicago, IL). A p <0.05 was considered significant.

Results

The clinical characteristics of the 139 early-stage breast cancer patients at the time of surgery are summarized in Table 1. Among these patients, the medium age was 49 years (ranging from 24 to 71 years). Histologically, all cases were invasive ductal carcinomas. Methylation of the BRCA1 promoter was detected in 78 (56%) of the 139 tumors examined in which 28 (20%) tumors were positive only for the methylated reaction and 50 (36%) were positive for both unmethylated and methylated reactions. Figure 1A shows representative methylation status of BRCA1 promoter by methylation-specific PCR. To evaluate the effect of DNA methylation on protein expression, immunohistochemical staining of BRCA1 was carried out. Consistent with the methylation status of BRCA1 promoter, the expression levels of BRCA1 protein were reduced in tumors with methylated genes (Figure 1B).

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Figure 1. BRCA1 promoter methylation and immunohistochemical analysis of BRCA1 in early-stage breast tumors.

(A) DNA methylation status of the BRCA1 promoter determined by methylation-specific PCR. M-labeled lanes represent PCR products amplified with methylation-specific primers (75 bp). U-labeled lanes indicate the presence of unmethylated genes (82 bp). Patients 1, 2, 3, 4, 6, and 7 show the presence of a PCR product in both reactions, indicating methylation of the BRCA1 promoter region. Patients 5 shows unmethylated gene. Molecular weight marker used is a 100-bp ladder. (B) Representative corresponding images of immunohistochemical staining of BRCA1. Scale bars, 100 µm.

https://doi.org/10.1371/journal.pone.0056256.g001

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Table 1. Patient clinical characteristics and BRCA1 promoter methylation in early-stage breast cancer.

https://doi.org/10.1371/journal.pone.0056256.t001

Chi-square analysis revealed that despite there was no significant difference between status of BRCA1 promoter methylation in age, tumor size, lymph node status, stage, histologic grade, ER, PR, and HER-2 positivity. A statistically significant association was found between BRCA1 promoter methylation and triple-negative breast cancer (ER-/PR-/HER2-) in this cohort of patients (p = 0.041). In Cox proportional hazards analysis, BRCA1 promoter methylation status was analyzed with tumor size, lymph node metastasis, histological grade, and age for their impact on OS and DFS. Adjusted for all these covariates, BRCA1 promoter methylation was identified as an independent predictor for OS (p = 0.027; hazard ratio = 16.38) and DFS (p = 0.003; hazard ratio = 12.19) (Table 2). No other clinical variable was found to be independently associated with death in this set of early-stage breast cancer patients. Kaplan-Meier survival curves showed that methylated promoter of the BRCA1 gene was associated with poor OS (p = 0.026; Figure 2A) and DFS (p = 0.001; Figure 2B).

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Figure 2. Kaplan-Meier curves of overall survival (A) and disease-free survival (B) for patients with early-stage breast cancer according to BRCA1 promoter methylation.

https://doi.org/10.1371/journal.pone.0056256.g002

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Table 2. Multivariate analysis of clinical factors and BRCA1 promoter methylation in patients with early-stage breast cancer for overall survival and disease-free survival.

https://doi.org/10.1371/journal.pone.0056256.t002

Discussion

The incidence of breast cancer in Taiwan has increased at a significantly faster pace than western countries over the last two decades [2] and stage 0 to IIA breast cancer currently comprises more than half of all newly diagnosed cases.

BRCA1 promoter hypermethylation has been implicated as one of the mechanisms of loss of gene expression and has been identified in 9–32% of unselected sporadic breast cancer [19], [25], [28][31]. We report here that hypermethylation of the BRCA1 gene promoter is present in 56% (78 of 139) of Taiwanese women with early-stage sporadic breast carcinomas, which is significantly higher than previously reported frequencies for this alteration in unselected sporadic breast tumors. The incidence of BRCA1 methylation has previously been reported to be higher in breast tumors of infiltrating ductal type [31]. Since all our patients are of the infiltrating ductal type, this finding was somewhat comparable to that of current literature.

BRCA1 protein expression was found to be absent or markedly decreased in the majority of the BRCA1 methylated tumors, suggesting epigenetic gene silencing in these tumors [31]. Breast cancers with BRCA1 promoter methylation also showed decreased expression of ER [25], [29], [31] and basal-like phenotype [25]. Our results indicated that BRCA1 promoter methylation correlated significantly with triple-negative breast cancer. (p = 0.041).

More than half of patients with BRCA1 mutation have triple-negative breast cancer, and share common clinical and pathological features [10], [32], [33]. However, a significant portion of triple-negative breast cancer patients do not carry BRCA1 mutations. Alteration in the function of the BRCA1 gene product has also been identified as an alternative route which also results in impaired BRCA1 function in triple-negative breast cancer [34]. Our finding suggests that BRCA1 promoter methylation may also have an etiological role on the development of triple-negative phenotype and underlie triple-negative breast cancer.

Given that BRCA1-associated breast cancers are more likely to be of high grade [35], or estrogen-receptor negative [35], and p53 positive [36], it has been speculated that BRCA1-related breast cancer is more aggressive than sporadic breast cancer. However, most studies conducted in specific populations suggest that survival for women with BRCA1 mutations is similar to that of women without the mutations [37], [38].

The Kaplan-Meier survival curves of the early-stage breast cancer patients plotted into 2 groups according methylation of BRCA1 promoter region showed that patients with methylated BRCA1 promoter had a significantly shorter OS and DFS than patients with unmethylated gene. (p = 0.026 and p = 0.001, respectively). Multivariate analysis adjusted for age, lymph node metastasis, size and grade of tumor also revealed that methylation of the BRCA1 promoter was an independent prognostic marker for survival in early-stage breast cancer (OS, p = 0.027; DFS, p = 0.003). Our findings suggests that BRCA1 promoter methylation is a better predictor of recurrence and survival than factors such as tumor size, lymph node metastasis, histological grade, and age in early-stage breast cancer.

In conclusion, this study highlights the frequent promoter methylation of BRCA1 and its prognostic significance, irrespective of BRCA1 gene mutation in Taiwanese patients with early-stage breast cancer. BRCA1 methylated tumors showed a statistically significant association with triple-negative status and exhibited poor outcome in terms of survival.

Genetic differences among different ethnicities/races may account for disparities in breast cancer susceptibility [3], [39]. Aberrant gene promoter methylation has also been shown to be affected by ethnicity in breast cancer [40]. Increased understanding of the genetic/epigenetic abnormality together with the ethnic factors/differences involved in the pathogenesis of breast cancer is crucial and may provide a basis for detection and treatment. Whether the phenomenon we observed in this study are due to ethnicity or etiology remained to be determine in larger studies that include breast cancer patients of different ethnicities/races.

Author Contributions

Conceived and designed the experiments: FC MH. Performed the experiments: NCH YH PC. Analyzed the data: NCH YH KKY. Wrote the paper: NCH.

References

  1. 1. (2012) Cancer Registry Annual Report, 2009 Taiwan. In: Health Do, editor.
  2. 2. Shen YC, Chang CJ, Hsu C, Cheng CC, Chiu CF, et al. (2005) Significant difference in the trends of female breast cancer incidence between Taiwanese and Caucasian Americans: implications from age-period-cohort analysis. Cancer Epidemiol Biomarkers Prev 14: 1986–1990.
  3. 3. Fong M, Henson DE, Devesa SS, Anderson WF (2006) Inter- and intra-ethnic differences for female breast carcinoma incidence in the continental United States and in the state of Hawaii. Breast Cancer Res Treat 97: 57–65.
  4. 4. Honrado E, Osorio A, Palacios J, Benitez J (2006) Pathology and gene expression of hereditary breast tumors associated with BRCA1, BRCA2 and CHEK2 gene mutations. Oncogene 25: 5837–5845.
  5. 5. Lynch HT, Silva E, Snyder C, Lynch JF (2008) Hereditary breast cancer: part I. Diagnosing hereditary breast cancer syndromes. Breast J 14: 3–13.
  6. 6. Chen W, Pan K, Ouyang T, Li J, Wang T, et al. (2009) BRCA1 germline mutations and tumor characteristics in Chinese women with familial or early-onset breast cancer. Breast Cancer Res Treat 117: 55–60.
  7. 7. Toh GT, Kang P, Lee SS, Lee DS, Lee SY, et al. (2008) BRCA1 and BRCA2 germline mutations in Malaysian women with early-onset breast cancer without a family history. PLoS One 3: e2024.
  8. 8. Ahn SH, Son BH, Yoon KS, Noh DY, Han W, et al. (2007) BRCA1 and BRCA2 germline mutations in Korean breast cancer patients at high risk of carrying mutations. Cancer Lett 245: 90–95.
  9. 9. Liede A, Narod SA (2002) Hereditary breast and ovarian cancer in Asia: genetic epidemiology of BRCA1 and BRCA2. Hum Mutat 20: 413–424.
  10. 10. Rennert G, Bisland-Naggan S, Barnett-Griness O, Bar-Joseph N, Zhang S, et al. (2007) Clinical outcomes of breast cancer in carriers of BRCA1 and BRCA2 mutations. N Engl J Med 357: 115–123.
  11. 11. Li SS, Tseng HM, Yang TP, Liu CH, Teng SJ, et al. (1999) Molecular characterization of germline mutations in the BRCA1 and BRCA2 genes from breast cancer families in Taiwan. Hum Genet 104: 201–204.
  12. 12. Chen ST, Chen RA, Kuo SJ, Chien YC (2003) Mutational screening of breast cancer susceptibility gene 1 from early onset, bi-lateral, and familial breast cancer patients in Taiwan. Breast Cancer Res Treat 77: 133–143.
  13. 13. Ford D, Easton DF, Stratton M, Narod S, Goldgar D, et al. (1998) Genetic heterogeneity and penetrance analysis of the BRCA1 and BRCA2 genes in breast cancer families. The Breast Cancer Linkage Consortium. Am J Hum Genet 62: 676–689.
  14. 14. Hedenfalk I, Ringner M, Ben-Dor A, Yakhini Z, Chen Y, et al. (2003) Molecular classification of familial non-BRCA1/BRCA2 breast cancer. Proc Natl Acad Sci U S A 100: 2532–2537.
  15. 15. Easton DF, Bishop DT, Ford D, Crockford GP (1993) Genetic linkage analysis in familial breast and ovarian cancer: results from 214 families. The Breast Cancer Linkage Consortium. Am J Hum Genet 52: 678–701.
  16. 16. Narod SA, Ford D, Devilee P, Barkardottir RB, Lynch HT, et al. (1995) An evaluation of genetic heterogeneity in 145 breast-ovarian cancer families. Breast Cancer Linkage Consortium. Am J Hum Genet 56: 254–264.
  17. 17. Baylin SB, Herman JG, Graff JR, Vertino PM, Issa JP (1998) Alterations in DNA methylation: a fundamental aspect of neoplasia. Adv Cancer Res 72: 141–196.
  18. 18. Jones PA, Laird PW (1999) Cancer epigenetics comes of age. Nat Genet 21: 163–167.
  19. 19. Esteller M, Silva JM, Dominguez G, Bonilla F, Matias-Guiu X, et al. (2000) Promoter hypermethylation and BRCA1 inactivation in sporadic breast and ovarian tumors. J Natl Cancer Inst 92: 564–569.
  20. 20. Matros E, Wang ZC, Lodeiro G, Miron A, Iglehart JD, et al. (2005) BRCA1 promoter methylation in sporadic breast tumors: relationship to gene expression profiles. Breast Cancer Res Treat 91: 179–186.
  21. 21. Rice JC, Massey-Brown KS, Futscher BW (1998) Aberrant methylation of the BRCA1 CpG island promoter is associated with decreased BRCA1 mRNA in sporadic breast cancer cells. Oncogene 17: 1807–1812.
  22. 22. Rice JC, Ozcelik H, Maxeiner P, Andrulis I, Futscher BW (2000) Methylation of the BRCA1 promoter is associated with decreased BRCA1 mRNA levels in clinical breast cancer specimens. Carcinogenesis 21: 1761–1765.
  23. 23. Wei M, Grushko TA, Dignam J, Hagos F, Nanda R, et al. (2005) BRCA1 promoter methylation in sporadic breast cancer is associated with reduced BRCA1 copy number and chromosome 17 aneusomy. Cancer Res 65: 10692–10699.
  24. 24. Barekati Z, Radpour R, Lu Q, Bitzer J, Zheng H, et al. (2012) Methylation signature of lymph node metastases in breast cancer patients. BMC Cancer 12: 244.
  25. 25. Bal A, Verma S, Joshi K, Singla A, Thakur R, et al. (2012) BRCA1-methylated sporadic breast cancers are BRCA-like in showing a basal phenotype and absence of ER expression. Virchows Arch 461: 305–312.
  26. 26. Krasteva ME, Bozhanov SS, Antov GG, Gospodinova ZI, Angelov SG (2012) Breast cancer patients with hypermethylation in the promoter of BRCA1 gene exhibit favorable clinical status. Neoplasma 59: 85–91.
  27. 27. Mirza S, Sharma G, Prasad CP, Parshad R, Srivastava A, et al. (2007) Promoter hypermethylation of TMS1, BRCA1, ERalpha and PRB in serum and tumor DNA of invasive ductal breast carcinoma patients. Life Sci 81: 280–287.
  28. 28. Xu CF, Brown MA, Chambers JA, Griffiths B, Nicolai H, et al. (1995) Distinct transcription start sites generate two forms of BRCA1 mRNA. Hum Mol Genet 4: 2259–2264.
  29. 29. Catteau A, Harris WH, Xu CF, Solomon E (1999) Methylation of the BRCA1 promoter region in sporadic breast and ovarian cancer: correlation with disease characteristics. Oncogene 18: 1957–1965.
  30. 30. Krop I, Maguire P, Lahti-Domenici J, Lodeiro G, Richardson A, et al. (2003) Lack of HIN-1 methylation in BRCA1-linked and "BRCA1-like" breast tumors. Cancer Res 63: 2024–2027.
  31. 31. Birgisdottir V, Stefansson OA, Bodvarsdottir SK, Hilmarsdottir H, Jonasson JG, et al. (2006) Epigenetic silencing and deletion of the BRCA1 gene in sporadic breast cancer. Breast Cancer Res 8: R38.
  32. 32. van der Groep P, Bouter A, van der Zanden R, Siccama I, Menko FH, et al. (2006) Distinction between hereditary and sporadic breast cancer on the basis of clinicopathological data. J Clin Pathol 59: 611–617.
  33. 33. Atchley DP, Albarracin CT, Lopez A, Valero V, Amos CI, et al. (2008) Clinical and pathologic characteristics of patients with BRCA-positive and BRCA-negative breast cancer. J Clin Oncol 26: 4282–4288.
  34. 34. Miyoshi Y, Murase K, Oh K (2008) Basal-like subtype and BRCA1 dysfunction in breast cancers. Int J Clin Oncol 13: 395–400.
  35. 35. Johannsson OT, Idvall I, Anderson C, Borg A, Barkardottir RB, et al. (1997) Tumour biological features of BRCA1-induced breast and ovarian cancer. Eur J Cancer 33: 362–371.
  36. 36. Sobol H, Stoppalyonnet D, Bressacdepaillerets B, Peyrat J, Guinebretiere J, et al. (1997) BRCA1-p53 relationship in hereditary breast cancer. Int J Oncol 10: 349–353.
  37. 37. Veronesi A, de Giacomi C, Magri MD, Lombardi D, Zanetti M, et al. (2005) Familial breast cancer: characteristics and outcome of BRCA 1–2 positive and negative cases. BMC Cancer 5: 70.
  38. 38. Verhoog LC, Brekelmans CT, Seynaeve C, van den Bosch LM, Dahmen G, et al. (1998) Survival and tumour characteristics of breast-cancer patients with germline mutations of BRCA1. Lancet 351: 316–321.
  39. 39. Shavers VL, Harlan LC, Stevens JL (2003) Racial/ethnic variation in clinical presentation, treatment, and survival among breast cancer patients under age 35. Cancer 97: 134–147.
  40. 40. Mehrotra J, Ganpat MM, Kanaan Y, Fackler MJ, McVeigh M, et al. (2004) Estrogen receptor/progesterone receptor-negative breast cancers of young African-American women have a higher frequency of methylation of multiple genes than those of Caucasian women. Clin Cancer Res 10: 2052–2057.