Neither targeted therapies nor predictors for chemotherapy sensitivity are available for triple-negative breast cancer (TNBC). Our study included 187 patients with TNBC, 164 of whom were treated with anthracycline-based adjuvant chemotherapy. Eleven molecular biomarkers were analyzed. BCL2, epidermal growth factor receptor (EGFR), MYC, TOP2A, and Ki-67 protein expression was evaluated by immunohistochemistry. The status of the EGFR, MYC, and TOP2A genes and chromosomes 7, 8, and 17 was assessed using fluorescence in situ hybridization. High BCL2 expression predicted poor relapse-free survival (RFS) in patients treated with anthracycline-based adjuvant chemotherapy (p = 0.035), poor breast cancer-specific survival (BCSS) (p = 0.048), and a trend to poor overall survival (OS) (p = 0.085). High levels of BCL2 expression predicted poor OS in basal-like (BL) TNBC patients treated with adjuvant anthracycline-based regimens (log-rank p = 0.033, hazard ratio (HR) 3.04, 95 % confidence interval (CI) 1.04–8.91) and a trend to poor RFS (log-rank p = 0.079) and poor BCSS (log-rank p = 0.056). Multivariate analysis showed that BCL2 status, tumor size, and nodal status all had independent predictive significance for RFS (p = 0.005, p = 0.091, p = 0.003, respectively; likelihood ratio test for the whole model, p = 0.003), BCSS (p = 0.012, p = 0.077, p = 0.01, respectively; likelihood ratio test for the whole model, p = 0.016), and OS (p = 0.008, p = 0.004, p = 0.004, respectively; likelihood ratio test for the whole model, p = 0.0006). Similarly, multivariate analysis for BL TNBC showed BCL2, tumor size, and nodal status all had independent predictive significance for RFS (likelihood ratio test for the whole model, p = 0.00125), BCSS (p = 0.00035), and OS (p = 0.00063). High EGFR expression was associated with poor BCSS (p = 0.039) in patients treated with anthracycline-based adjuvant chemotherapy. Patients who underwent anthracycline-based adjuvant chemotherapy and exhibited CMYC amplification had a trend to worse BCSS (p = 0.066). In conclusion, high BCL2 expression is a significant independent predictor of poor outcome in TNBC patients treated with anthracycline-based adjuvant chemotherapy, and this is the first study showing the BCL2 prediction in BL TNBC. BCL2 expression analysis could facilitate decision making on adjuvant treatment in TNBC patients.
Adjuvant chemotherapy Anthracycline BCL2 EGFR Predictive marker Triple-negative breast cancer
This is a preview of subscription content, log in to check access
We thank Jiri Bartek and Alice Hlobilkova for many helpful comments. We also thank Dana Knoflickova, Sona Mlcochova, and Eva Sedlakova for excellent technical assistance.
This work was supported by the Czech Ministry of Health (IGA NS10286-3, IGA NS10357-3, IGA NT14599-32013). The infrastructural part of the project (Institute of Molecular and Translational Medicine, Biomedreg) was supported under the Operational Programme Research and Development for Innovations (CZ.1.05/2.1.00/01.0030), National Sustainability Programme (LO1304), and Czech Technology Agency (Center of Competence for Molecular Diagnostics and Personalized Medicine) (TE02000058).
Conflicts of interest
MH and RT are owners of Intellmed stock.
KB conceived, designed, and directed the study; analyzed the data; and wrote the manuscript. MS obtained clinical data. GK performed IHC (BCL2, TOP2A, EGFR, CMYC), and JV and LR performed the statistical analyses. JB had oversight of the study’s progression and contributed to the data analysis and the writing of the manuscript. RT and VK performed the FISH experiments. KC and MH contributed to the editing of the manuscript. ZK contributed to the writing of the manuscript. All authors approved the manuscript.
Lehmann BD, Bauer JA, Chen X, Sanders ME, Chakravarthy AB, Shyr Y, et al. Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies. J Clin Invest. 2011;121:2750–67.CrossRefPubMedPubMedCentralGoogle Scholar
Rody A, Karn T, Liedtke C, Pusztai L, Ruckhaeberle E, Hanker L, et al. A clinically relevant gene signature in triple negative and basal-like breast cancer. Breast Cancer Res. 2011;13:R97.CrossRefPubMedPubMedCentralGoogle Scholar
Shah SP, Roth A, Goya R, Oloumi A, Ha G, Zhao Y, et al. The clonal and mutational evolution spectrum of primary triple-negative breast cancers. Nature. 2012;486:395–9.PubMedGoogle Scholar
Stephens PJ, McBride DJ, Lin ML, Varela I, Pleasance ED, Simpson JT, et al. Complex landscapes of somatic rearrangement in human breast cancer genomes. Nature. 2009;462:1005–10.CrossRefPubMedPubMedCentralGoogle Scholar
Silver DP, Richardson AL, Eklund AC, Wang ZC, Szallasi Z, Li Q, et al. Efficacy of neoadjuvant cisplatin in triple-negative breast cancer. J Clin Oncol. 2010;28:1145–53.CrossRefPubMedPubMedCentralGoogle Scholar
Eiermann W, Bergh J, Cardoso F, Conte P, Crown J, Curtin NJ, et al. Triple negative breast cancer: proposals for a pragmatic definition and implications for patient management and trial design. Breast. 2012;21:20–6.CrossRefPubMedGoogle Scholar
Masuda H, Baggerly KA, Wang Y, Zhang Y, Gonzalez-Angulo AM, Meric-Bernstam F, et al. Differential response to neoadjuvant chemotherapy among 7 triple-negative breast cancer molecular subtypes. Clin Cancer Res. 2013;19:5533–40.CrossRefPubMedGoogle Scholar
Dawson SJ, Makretsov N, Blows FM, Driver KE, Provenzano E, Le QJ, et al. BCL2 in breast cancer: a favourable prognostic marker across molecular subtypes and independent of adjuvant therapy received. Br J Cancer. 2010;103:668–75.CrossRefPubMedPubMedCentralGoogle Scholar
Basu A, Haldar S. The relationship between BcI2, Bax and p53: consequences for cell cycle progression and cell death. Mol Hum Reprod. 1998;4:1099–109.CrossRefPubMedGoogle Scholar
Pusztai L, Krishnamurti S, Perez CJ, Sneige N, Esteva FJ, Volchenok M, et al. Expression of BAG-1 and BcL-2 proteins before and after neoadjuvant chemotherapy of locally advanced breast cancer. Cancer Investig. 2004;22:248–56.CrossRefGoogle Scholar
Dickinson PD, Abdel-Fatah TMA, Green AR et al. Bcl2 expression and prediction of outcomes to anthracycline-based neoadjuvant chemotherapy in ER-positive breast cancer and to nonanthracycline adjuvant therapy. J Clin Oncol 2011;29:(suppl; abstr 589).Google Scholar
Abdel-Fatah TM, Perry C, Dickinson P, Ball G, Moseley P, Madhusudan S, et al. Bcl2 is an independent prognostic marker of triple negative breast cancer (TNBC) and predicts response to anthracycline combination (ATC) chemotherapy (CT) in adjuvant and neoadjuvant settings. Ann Oncol. 2013;24:2801–7.CrossRefPubMedGoogle Scholar
Hynes NE, Lane HA. ERBB receptors and cancer: the complexity of targeted inhibitors. Nat Rev Cancer. 2005;5:341–54.CrossRefPubMedGoogle Scholar
Hobday TJ, Perez EA. Molecularly targeted therapies for breast cancer. Cancer Control. 2005;12:73–81.PubMedGoogle Scholar
Bouchalova K, Cizkova M, Cwiertka K, Trojanec R, Hajduch M. Triple negative breast cancer–current status and prospective targeted treatment based on HER1 (EGFR), TOP2A and C-MYC gene assessment. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2009;153:13–7.CrossRefPubMedGoogle Scholar
Viale G, Rotmensz N, Maisonneuve P, Bottiglieri L, Montagna E, Luini A, et al. Invasive ductal carcinoma of the breast with the “triple-negative” phenotype: prognostic implications of EGFR immunoreactivity. Breast Cancer Res Treat. 2009;116:317–28.CrossRefPubMedGoogle Scholar
Liu D, He J, Yuan Z, Wang S, Peng R, Shi Y, et al. EGFR expression correlates with decreased disease-free survival in triple-negative breast cancer: a retrospective analysis based on a tissue microarray. Med Oncol. 2012;29:401–5.CrossRefPubMedGoogle Scholar
Jarvinen TA, Tanner M, Rantanen V, Barlund M, Borg A, Grenman S, et al. Amplification and deletion of topoisomerase IIalpha associate with ErbB-2 amplification and affect sensitivity to topoisomerase II inhibitor doxorubicin in breast cancer. Am J Pathol. 2000;156:839–47.CrossRefPubMedPubMedCentralGoogle Scholar
Press MF, Sauter G, Buyse M, Bernstein L, Guzman R, Santiago A, et al. Alteration of topoisomerase II-alpha gene in human breast cancer: association with responsiveness to anthracycline-based chemotherapy. J Clin Oncol. 2011;29:859–67.CrossRefPubMedGoogle Scholar
Zaczek AJ, Markiewicz A, Seroczynska B, Skokowski J, Jaskiewicz J, Pienkowski T, et al. Prognostic significance of TOP2A gene dosage in HER-2-negative breast cancer. Oncologist. 2012;17:1246–55.CrossRefPubMedPubMedCentralGoogle Scholar
Ali HR, Dawson SJ, Blows FM, Provenzano E, Leung S, Nielsen T, et al. A Ki67/BCL2 index based on immunohistochemistry is highly prognostic in ER-positive breast cancer. J Pathol. 2012;226:97–107.CrossRefPubMedGoogle Scholar
Munzone E, Botteri E, Sciandivasci A, Curigliano G, Nole F, Mastropasqua M, et al. Prognostic value of Ki-67 labeling index in patients with node-negative, triple-negative breast cancer. Breast Cancer Res Treat. 2012;134:277–82.CrossRefPubMedGoogle Scholar
McShane LM, Altman DG, Sauerbrei W, Taube SE, Gion M, Clark GM. REporting recommendations for tumor MARKer prognostic studies (REMARK). Nat Clin Pract Oncol. 2005;2:416–22.CrossRefPubMedGoogle Scholar
Kolar Z, Murray PG, Scott K, Harrison A, Vojtesek B, Dusek J. Relation of Bcl-2 expression to androgen receptor, p21WAF1/CIP1, and cyclin D1 status in prostate cancer. Mol Pathol. 2000;53:15–8.CrossRefPubMedPubMedCentralGoogle Scholar
Bouchalova K, Trojanec R, Kolar Z, Cwiertka K, Cernakova I, Mihal V, et al. Analysis of ERBB2 and TOP2A gene status using fluorescence in situ hybridization versus immunohistochemistry in localized breast cancer. Neoplasma. 2006;53:393–401.PubMedGoogle Scholar
Nielsen TO, Hsu FD, Jensen K, Cheang M, Karaca G, Hu Z, et al. Immunohistochemical and clinical characterization of the basal-like subtype of invasive breast carcinoma. Clin Cancer Res. 2004;10:5367–74.CrossRefPubMedGoogle Scholar
R Development Core Team (2011). R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL http://www.R-project.org/.
Bouchalova K, Svoboda M, Kharaishvili G, Radova L, Bouchal J, Trojanec R, et al. BCL2 protein in prediction of relapse in triple-negative breast cancer (TNBC) treated with adjuvant anthracycline-based chemotherapy. J Clin Oncol. 2012;30:suppl; abstr 1087.Google Scholar
Abdel-Fatah TMA, Dickinson PD, Moseley P et al. Bcl2 as a surrogate prognostic and predictive marker in triple-negative breast cancer. J Clin Oncol 2011;29:(suppl; abstr 1024).Google Scholar
Moulder SL, Symmans WF, Booser DJ, Madden TL, Lipsanen C, Yuan L, et al. Phase I/II study of G3139 (Bcl-2 antisense oligonucleotide) in combination with doxorubicin and docetaxel in breast cancer. Clin Cancer Res. 2008;14:7909–16.CrossRefPubMedPubMedCentralGoogle Scholar
Bouchalova K, Cizkova M, Trojanec R et al. BCL2 is a predictive marker of adjuvant CMF regimen in triple negative breast cancer (TNBC) patients. Breast Cancer Research 2011;13:(suppl 2; P8; doi: 10.1186/bcr3029).
Bozojevic-Spasojevic I, Ameye L, Paesmans M, Larsimont D, Di Leo A, Dolci S, et al. Prognostic, predictive abilities and concordance of BCL2 and TP53 protein expression in primary breast cancers and axillary lymph nodes: a retrospective analysis of the Belgian three arm study evaluating anthracycline vs CMF adjuvant chemotherapy. Breast. 2014;23:473–81.CrossRefGoogle Scholar
Choi JE, Kang SH, Lee SJ, Bae YK. Prognostic significance of Bcl-2 expression in non-basal triple-negative breast cancer patients treated with anthracycline-based chemotherapy. Tumor Biol. 2014;35:12255–63.CrossRefGoogle Scholar
Bouchalova K, Kharaishvili G, Bouchal J, Vrbkova J, Megova M, Hlobilkova A. Triple negative breast cancer - BCL2 in prognosis and prediction. Review. Current Drug Targets. 2014;15(12):1166–75.CrossRefPubMedGoogle Scholar
Horiuchi D, Kusdra L, Huskey NE, Chandriani S, Lenburg ME, Gonzalez-Angulo AM, et al. MYC pathway activation in triple-negative breast cancer is synthetic lethal with CDK inhibition. J Exp Med. 2012;209:679–96.CrossRefPubMedPubMedCentralGoogle Scholar
Knoop AS, Knudsen H, Balslev E, Rasmussen BB, Overgaard J, Nielsen KV, et al. Retrospective analysis of topoisomerase IIa amplifications and deletions as predictive markers in primary breast cancer patients randomly assigned to cyclophosphamide, methotrexate, and fluorouracil or cyclophosphamide, epirubicin, and fluorouracil: Danish Breast Cancer Cooperative Group. J Clin Oncol. 2005;23:7483–90.CrossRefPubMedGoogle Scholar
Gunnarsdottir KA, Jensen MB, Zahrieh D, Gelber RD, Knoop A, Bonetti M, et al. CEF is superior to CMF for tumours with TOP2A aberrations: a Subpopulation Treatment Effect Pattern Plot (STEPP) analysis on Danish Breast Cancer Cooperative Group Study 89D. Breast Cancer Res Treat. 2010;123:163–9.CrossRefPubMedPubMedCentralGoogle Scholar
Nielsen KV, Muller S, Moller S, Schonau A, Balslev E, Knoop AS, et al. Aberrations of ERBB2 and TOP2A genes in breast cancer. Mol Oncol. 2010;4:161–8.CrossRefPubMedGoogle Scholar
Pohl G, Rudas M, Taucher S, Stranzl T, Steger GG, Jakesz R, et al. Expression of cell cycle regulatory proteins in breast carcinomas before and after preoperative chemotherapy. Breast Cancer Res Treat. 2003;78:97–103.CrossRefPubMedGoogle Scholar
Chang J, Ormerod M, Powles TJ, Allred DC, Ashley SE, Dowsett M. Apoptosis and proliferation as predictors of chemotherapy response in patients with breast carcinoma. Cancer. 2000;89:2145–52.CrossRefPubMedGoogle Scholar
Petit T, Wilt M, Velten M, Millon R, Rodier JF, Borel C, et al. Comparative value of tumour grade, hormonal receptors, Ki-67, HER-2 and topoisomerase II alpha status as predictive markers in breast cancer patients treated with neoadjuvant anthracycline-based chemotherapy. Eur J Cancer. 2004;40:205–11.CrossRefPubMedGoogle Scholar
Desmedt C, Di Leo A, Larsimont D, Haibe-Kains B, Selleslags J, et al. Multifactorial approach to predicting resistance to anthracyclines. J Clin Oncol. 2011;29:1578–86.CrossRefPubMedGoogle Scholar
Dent R, Trudeau M, Pritchard KI, Hanna WM, Kahn HK, Sawka CA, et al. Triple-negative breast cancer: clinical features and patterns of recurrence. Clin Cancer Res. 2007;13:4429–34.CrossRefPubMedGoogle Scholar