Breast Cancer Research and Treatment

, Volume 127, Issue 1, pp 179–193 | Cite as

Prognostic utility of β-tubulin isotype III and correlations with other molecular and clinicopathological variables in patients with early breast cancer: a translational Hellenic Cooperative Oncology Group (HeCOG) study

  • George Pentheroudakis
  • Anna Batistatou
  • Konstantine T. Kalogeras
  • Ralf Kronenwett
  • Ralph M. Wirtz
  • Evangelos Bournakis
  • Anastasia G. Eleftheraki
  • Dimitrios Pectasides
  • Mattheos Bobos
  • Irini Papaspirou
  • Sevasti Kamina
  • Helen Gogas
  • Angelos K. Koutras
  • Nicholas Pavlidis
  • George Fountzilas
Clinical trial


We evaluated the prognostic and predictive utility of β-tubulin isotype III (TUBB3) tumour gene transcription in early breast cancer patients enrolled in a randomised study. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was applied for assessment of TUBB3, ER, PgR, HER2 and MAPT messenger RNA and immunohistochemistry (IHC) for protein expression in 314 patients enrolled in trial HE10/97, evaluating epirubicin–alkylator adjuvant chemotherapy with or without paclitaxel. High TUBB3 mRNA status was associated with advanced T stage, high histological grade, low mRNA and protein levels of ER, PgR and MAPT, and high levels of HER2 (p < 0.001). At a median follow-up of 98 months, multivariate analysis showed high TUBB3 mRNA status to have prognostic significance for DFS (HR = 1.83, 95% CI 1.25–2.68, p = 0.002) and OS (HR = 1.71, 95% CI 1.03–2.83, p = 0.038), along with the number of involved axillary nodes, PgR mRNA status and tumour grade. TUBB3 mRNA levels did not predict benefit from inclusion of paclitaxel in adjuvant chemotherapy (test for interaction p = 0.96 for OS, p = 0.46 for DFS). Transcriptional activity of β-tubulin isotype III in early breast cancer is an adverse prognostic factor, though not a predictive one for taxane efficacy.


Breast cancer β-tubulin isotype III Adjuvant chemotherapy Prognostic/predictive factors 



The authors wish to thank Ms. Evita Fragou and Ms. Dimitra Katsala for monitoring the study, Ms. Maria Moschoni for coordinating the data management and Ms. Thalia Spinari for tissue sample collection.


  1. 1.
    EBCTC Group (2005) Effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year survival:an overview of the randomised trials. Lancet 365:1687–1717CrossRefGoogle Scholar
  2. 2.
    Trudeau M, Charbonneau F, Gelmon K et al (2005) Selection of adjuvant chemotherapy for treatment of node-positive breast cancer. Lancet Oncol 6:886–898PubMedCrossRefGoogle Scholar
  3. 3.
    Bedard P, Leo A, Piccart-Gebhart M (2010) Taxanes: optimizing adjuvant chemotherapy for early-stage breast cancer. Nat Rev Clin Oncol 7:22–36PubMedCrossRefGoogle Scholar
  4. 4.
    Bhat KMR, Setaluri V (2007) Microtubule-associated proteins as targets in cancer chemotherapy. Clin Cancer Res 13:2849–2854PubMedCrossRefGoogle Scholar
  5. 5.
    Mollinedo F, Gajate C (2003) Microtubules, microtubule-interfering agents and apoptosis. Apoptosis 8:413–450PubMedCrossRefGoogle Scholar
  6. 6.
    Kavallaris M (2010) Microtubules and resistance to tubulin-binding agents. Nat Rev Cancer 10:194–204PubMedCrossRefGoogle Scholar
  7. 7.
    Fountzilas G, Skarlos D, Dafni U, Gogas H, Briasoulis E et al (2005) Postoperative dose-dense sequential chemotherapy with epirubicin, followed byCMFwith or without paclitaxel, in patients with high-risk operable breast cancer: a randomised phase III study conducted by the Hellenic Cooperative Oncology Group. Ann Oncol 16:1762–1771PubMedCrossRefGoogle Scholar
  8. 8.
    Bohmann K, Hennig G, Rogel U, Poremba C, Mueller BM, Fritz P, Stoerkel S, Schaefer K-L (2009) RNA extraction from archival formalin-fixed paraffin-embedded tissue: a comparison of manual, semiautomated, and fully automated purification methods. Clin Chem 55:1719–1727PubMedCrossRefGoogle Scholar
  9. 9.
    Mueller BM, Kronenwett R, Hennig G, Euting H, Weber K et al (2011) Quantitative determination of estrogen receptor, progesterone receptor and HER2 mRNA in formalin-fixed paraffin-embedded tissue—a new option for predictive biomarker assessment in breast cancer. Diagn Mol Pathol 20:1–10CrossRefGoogle Scholar
  10. 10.
    Hammond ME, Hayes DF, Dowsett M, Allred DC, Hagerty KL, Badve S et al (2010) American Society of Clinical Oncology/College Of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer. J Clin Oncol 28:2784–2795PubMedCrossRefGoogle Scholar
  11. 11.
    Wolff AC, Hammond ME, Schwartz JN, Hagerty KL, Allred DC, Cote RJ et al (2007) American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. Arch Pathol Lab Med 131:18–43PubMedGoogle Scholar
  12. 12.
    Pusztai L, Jeong J-H, Gong Y, Ross JS, Kim C, Paik S, et al (2009) Evaluation of microtubule-associated protein-tau expression as a prognostic and predictive marker in the NSABP-B 28 randomized clinical trial. J Clin Oncol 27:4287–4292PubMedCrossRefGoogle Scholar
  13. 13.
    Pentheroudakis G, Kalogeras K, Wirtz R, Grimani I, Zografos G, Gogas H et al (2009) Gene expression of estrogen receptor, progesterone receptor and microtubule-associated protein Tau in high-risk early breast cancer: a quest for molecular predictors of treatment benefit in the context of a Hellenic Cooperative Oncology Group trial. Breast Cancer Res Treat 116:131–143PubMedCrossRefGoogle Scholar
  14. 14.
    Hudis TL (2007) Proposal for standardized definitions for efficacy end points in adjuvant breast cancer trials: the STEEP system. J Clin Oncol 25:2127–2132PubMedCrossRefGoogle Scholar
  15. 15.
    McShane LM, Altman DG, Sauerbrei W et al (2005) Reporting recommendations for tumor marker prognostic studies. J Clin Oncol 23:9067–9072PubMedCrossRefGoogle Scholar
  16. 16.
    Jordan MA, Wilson L (2004) Microtubules as a target for anticancer drugs. Nat Rev Cancer 4:253–265PubMedCrossRefGoogle Scholar
  17. 17.
    Cleveland DW, Sullivan KF (1985) Molecular biology and genetics of tubulin. Annu Rev Biochem 54:331–365PubMedCrossRefGoogle Scholar
  18. 18.
    Lopata MA, Cleveland DW (1987) In vivo microtubules are copolymers of available h-tubulin isotypes: localization of each of six vertebrate h-tubulin isotypes using polyclonal antibodies elicited by synthetic peptide antigens. J Cell Biol 105:1707–1720PubMedCrossRefGoogle Scholar
  19. 19.
    Cicchillitti L, Penci R, Di Michele M, Fillipetti F, Rotilio D, Benedetta Donati M et al (2008) Proteomic characterisation of cytoskeletal and mitochondrial class III b-tubulin. Mol Cancer Ther 7:2070–2079PubMedCrossRefGoogle Scholar
  20. 20.
    Orr GA, Verdier-Pinard P, McDaid H, Horwitz SB (2003) Mechanisms of taxol resistance related to microtubules. Oncogene 22:7280–7295PubMedCrossRefGoogle Scholar
  21. 21.
    Mozzetti S, Ferlini C, Concolino P, Fillippetti F, Raspaglio G, Prislei S et al (2005) Class III b-tubulin overexpression is a prominent mechanism of paclitaxel resistance in ovarian cancer patients. Clin Cancer Res 11:298–305PubMedGoogle Scholar
  22. 22.
    Hasegawa S, Miyoshi Y, Egawa C et al (2003) Prediction of response to docetaxel by quantitative analysis of class I and III beta-tubulin isotype mRNA expression in human breast cancers. Clin Cancer Res 9:2992–2997PubMedGoogle Scholar
  23. 23.
    Magnani M, Ortuso F, Soro S, Alcaro S, Tramontano A, Botta M (2006) The beta/beta III tubulin isoforms and their complexes with antimitotic agents: Docking and molecular dynamics studies. FEBS J 273:3301–3310PubMedCrossRefGoogle Scholar
  24. 24.
    Seve P, Dumontet C (2008) Is class III b-tubulin a predictive factor in patients receiving tubulin-binding agents? Oncology 9:168–175PubMedGoogle Scholar
  25. 25.
    Seve P, Lai R, Ding K et al (2007) Class III beta tubulin expression and benefit from adjuvant cisplatin/vinorelbine chemotherapy in operable non-small cell lung cancer. Analysis of NCIC BR.10. Clin Cancer Res 13:994–999PubMedCrossRefGoogle Scholar
  26. 26.
    Dumontet C, Krajewska M, Treilleux I, Mackey JR, Martin M, Rupin M et al (2010) BCIRG 001 molecular analysis: prognostic factors in node-positive breast cancer patients receiving adjuvant chemotherapy. Clin Cancer Res 16:3988–3997PubMedCrossRefGoogle Scholar
  27. 27.
    Dennis K, Ulttenbogaard M, Chiaramello A, Moody SA (2002) Cloning and characterisation of 5-flanking region of the rat neuron-specific class III beta-tubulin gene. Gene 294:269–277PubMedCrossRefGoogle Scholar
  28. 28.
    Raspaglio G, Filipetti F, Prislei S et al (2008) Hypoxia induces Class III beta-tubulin gene expression by HIF1a binding to its 3-flanking region. Gene 409:100–108PubMedCrossRefGoogle Scholar
  29. 29.
    Gan PP, Pasquier E, Kavallaris M (2007) Class III b-tubulin mediates sensitivity to chemotherapeutic drugs in non small cell lung cancer. Cancer Res 67:9356–9363PubMedCrossRefGoogle Scholar
  30. 30.
    Loo LW, Grove DI, Williams EM, Neal CL, Cousens LA et al (2004) Array comparative genomic hybridization analysis of genomic alterations in breast cancer subtypes. Cancer Res 64(23):8541–8549PubMedCrossRefGoogle Scholar
  31. 31.
    Kaur GP, Reddy DE, Zimonjic DB, de Riel JK, Athwal RS (2005) Functional identification of a BAC clone from 16q24 carrying a senescence gene SEN16 for breast cancer cells. Oncogene 24(1):47–54PubMedCrossRefGoogle Scholar
  32. 32.
    Chin SF, Wang Y, Thorne NP, Teschendorff AE, Pinder SE, Vias M et al (2007) Using array-comparative genomic hybridization to define molecular portraits of primary breast cancers. Oncogene 26(13):1959–1970PubMedCrossRefGoogle Scholar
  33. 33.
    Mongroo PS, Rustgi AK (2010) The role of the miR-200 family in epithelial-mesenchymal transition. Cancer Biol Ther 10(3):219–222PubMedCrossRefGoogle Scholar
  34. 34.
    Goldstein NS, Ferkowicz M, Odish E et al (2003) Minimum formalin fixation time for consistent estrogen receptor immunocytochemical staining of invasive breast carcinoma. Am J Clin Pathol 120:86–92PubMedCrossRefGoogle Scholar
  35. 35.
    Rhodes A (2003) Quality assurance in immunohistochemistry. Am J Surg Pathol 27:1284–1285PubMedGoogle Scholar
  36. 36.
    Morris KV (2008) RNA-mediated transcriptional gene silencing in human cells. Curr Top Microbiol Immunol 320:211–224PubMedCrossRefGoogle Scholar
  37. 37.
    Ravo M, Mutarelli M, Ferraro L, Grober OM, Paris O et al (2008) Quantitative expression profiling of highly degraded RNA from formalin-fixed paraffin-embedded breast tumour biopsies by oligonucleotide microarrays. Lab Invest 88:430–440PubMedCrossRefGoogle Scholar
  38. 38.
    Yasuda J, Hayashizaki Y (2008) The RNA continent. Adv Cancer Res 99:77–112PubMedCrossRefGoogle Scholar
  39. 39.
    Rouzier R, Rajan R, Wagner P, Hess KR, Gold DL et al (2005) Microtubule-associated protein tau: a marker of paclitaxel sensitivity in breast cancer. PNAS 102:8315–8320PubMedCrossRefGoogle Scholar
  40. 40.
    Andre F, Hatzis C, Anderson K, Sotiriou S, Mazouni C et al (2007) Microtubule-associated protein tau is a bifunctional predictor of endocrine sensitivity and chemotherapy resistance in estrogen-receptor positive breast cancer. Clin Cancer Res 13:2061–2067PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2011

Authors and Affiliations

  • George Pentheroudakis
    • 1
  • Anna Batistatou
    • 2
  • Konstantine T. Kalogeras
    • 3
    • 4
  • Ralf Kronenwett
    • 5
    • 13
  • Ralph M. Wirtz
    • 5
    • 14
  • Evangelos Bournakis
    • 6
  • Anastasia G. Eleftheraki
    • 7
  • Dimitrios Pectasides
    • 8
  • Mattheos Bobos
    • 9
  • Irini Papaspirou
    • 10
  • Sevasti Kamina
    • 2
  • Helen Gogas
    • 11
  • Angelos K. Koutras
    • 12
  • Nicholas Pavlidis
    • 1
  • George Fountzilas
    • 3
  1. 1.Department of Medical OncologyIoannina University HospitalIoanninaGreece
  2. 2.Department of PathologyIoannina University HospitalIoanninaGreece
  3. 3.Department of Medical OncologyPapageorgiou Hospital, Aristotle University of Thessaloniki School of MedicineThessalonikiGreece
  4. 4.Translational Research SectionHellenic Cooperative Oncology Group, Data OfficeAthensGreece
  5. 5.Siemens Healthcare DiagnosticsCologneGermany
  6. 6.Department of Clinical TherapeuticsAlexandra Hospital, University of Athens School of MedicineAthensGreece
  7. 7.Section of BiostatisticsHellenic Cooperative Oncology Group, Data OfficeAthensGreece
  8. 8.Section of Oncology, 2nd Department of Internal MedicineHippokration Hospital, University of Athens School of MedicineAthensGreece
  9. 9.Laboratory of Molecular Oncology, Hellenic Foundation for Cancer ResearchAristotle University of Thessaloniki School of MedicineThessalonikiGreece
  10. 10.Department of HistopathologyAlexandra HospitalAthensGreece
  11. 11.1st Department of MedicineLaiko General Hospital, University of Athens School of MedicineAthensGreece
  12. 12.Division of Oncology, Department of MedicineUniversity Hospital, University of Patras Medical SchoolPatrasGreece
  13. 13.Sividon Diagnostics GmbHCologneGermany
  14. 14.Stratifyer Molecular Pathology GmbHCologneGermany

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