DOI:10.1093/jnci/dju094 First published online May 9, 2014
© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: [email protected].
Editorial
Brachyury: A New Player in Promoting Breast Cancer Aggressiveness Maira M. Pires, Stuart A. Aaronson Correspondence to: Stuart A. Aaronson, MD, Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Pl, Box 1130, New York, NY 10029 (e-mail: [email protected]).
1 of 2 Editorial | JNCI
cells to invade the extracellular matrix, 2) increased primary and secondary mammosphere formation, and 3) increased resistance to chemotherapy. The authors also sought to determine whether markers of stemness were altered by brachyury. Indeed, SOX2, NANOG and OCT4 were directly modulated by overexpression or downregulation of brachyury. Thus, Palena et al. (26) show for the first time that a member of the T-box TF family of proteins modulates these important pluripotency TFs. In comparing brachyury expression in normal and BC samples, Palena et al. (26) found that, whereas 21% of the 118 breast carcinoma samples analyzed were positive for brachyury mRNA expression by quantitative real-time polymerase chain reaction, approximately 90% of a different set of 30 primary breast tumors were positive by immunohistochemistry. Among 16 benign breast tissues examined, only two samples, both fibroadenomas, were positive by immunohistochemistry for this T-box protein. Further studies comparing the sensitivity of these approaches on the same samples should clarify the basis for the differences observed. For example, immunohistochemistry, which has been historically more difficult to quantitate (28,29), may overestimate the frequency of brachyury detection in BCs. Nonetheless, Palena et al.’s evidence associating brachyury with in vitro BC invasiveness, upregulation of stem cell TFs, and higher recurrence and metastatic spread suggests that brachyury could provide a new marker for aggressive breast tumors (26). Palena et al. (26) also developed a vector-based vaccine expressing brachyury aimed at targeting and killing brachyury-positive breast carcinoma cells to evaluate the possibility of a therapeutic application in BC patients. In fact, this vaccine, which uses a 9-mer peptide of brachyury that binds to human leukocyte antigen-A2, was shown previously to have efficacy in targeting other brachyury-positive tumor cells in vitro, such as lung cancer cells (30–33). Because their results demonstrated evidence of killing brachyurypositive BC cell lines in vitro, the authors argue that a brachyury vaccine approach could be considered as either mono- or combined therapy for the subset of BCs expressing this protein (26). In fact, a proposed brachyury-targeted vaccine is now being evaluated in a phase I clinical trial designed to establish safety and tolerability (34). Some questions raised by the work of Palena et al. also warrant further investigation. Although there was an indication that brachyury mRNA levels were higher in BCs negative for estrogen and progesterone receptors, brachyury was not correlated with a specific subtype of BC. Further studies encompassing larger numbers of BC samples will undoubtedly address whether brachyury Vol. 106, Issue 5 | dju094 | May 14, 2014
Downloaded from http://jnci.oxfordjournals.org/ at Univ of Iowa-Law Library on May 23, 2015
In the United States, breast cancer (BC) in women is the second most frequent cause of death and the second most diagnosed cancer, leading to approximately 230 000 new cases of invasive BC and approximately 40 000 new deaths a year (1,2). A major unmet need for all BCs is better understanding of what drives invasiveness and metastasis, factors responsible for the lethality of this disease. Epithelial-to-mesenchymal transition (EMT), initially described as a critical process in embryogenesis (3) and wound healing (4–7), has been recognized as a possible trigger for metastatic progression of tumors (4,8,9). This important and sometimes reversible process is associated with loss of cell-to-cell adhesion, increased cell motility and invasive properties, and specific morphogenetic changes (8,10). During EMT, certain epithelial markers, such as E-cadherin, are decreased or lost, whereas mesenchymal markers, such as N-cadherin, vimentin, snail, slug, and twist, are upregulated (4,8,10). The latter three are transcription factors (TFs) that are known to control EMT events associated with cancer progression. Brachyury, first discovered in 1927 (11), is the founding member of the highly conserved T-box TF family, whose members share a common DNA-binding domain of 180 to 200 amino acids known as the T-domain (12). Varying degrees of homology between the T-domain of T-box genes subdivide the family into subfamilies: Brachyury, T-brain I, Tbx1, Tbx2, and Tbx6. Within each subfamily, there may be multiple T-box genes (12). Over the past few years, several members of the T-box subfamilies, including brachyury, have been implicated in EMT and cancer. For example, in the T-brain I subfamily, eomesodermin has been reported to be not only involved in developmental EMT (13) but also in lymph node metastasis (14). Within the Tbx1 subfamily, both tbx18 and tbx20 can control different aspects of developmental EMT (15,16). In the Tbx2 subfamily, both tbx2 and tbx3 have been implicated in driving EMT and being overexpressed in cancers such as BCs (17–25). In this issue of the Journal, a new study by Palena et al. (26) focuses on the role of brachyury in BC. This study classified more than 4000 breast tumor samples for their levels of brachyury mRNA expression from 23 published databases from the Gene Expression Omnibus (27). After brachyury levels were subgrouped into low, intermediate, and high expression, Kaplan–Meier analysis was performed on a set of 357 BC patients treated with adjuvant tamoxifen monotherapy for 5 years to assess prognostic differences among the three subgroups. High brachyury mRNA was associated with higher risk of recurrence and distant metastasis. Furthermore, exogenous modulation of brachyury expression in BC cells revealed that higher brachyury levels 1) enhanced the ability of such BC
overexpression is more specific to a particular subtype of BC or whether it is a general marker for BC metastasis and poor survival. Furthermore, mechanistic dissection of brachyury downstream target genes responsible for mediating its invasive effects could prove valuable in identifying future therapeutic targets. Brachyury is now the third member of the T-box proteins, along with tbx2 and tbx3, to be implicated in driving various aspects of BC biology, such as induction of EMT and invasiveness. Palena et al.’s findings (26) contribute to our knowledge of a new driver of the metastatic phenotype of BC cells and suggest that future investigations of other less well-characterized T-box protein family members should provide a more complete picture of the importance of T-box TFs in driving BC and other carcinomas. References
jnci.oxfordjournals.org
NCT01519817. Accessed March 27, 2014.
Notes The authors have no conflicts of interest to declare. Affiliation of authors: Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY (MMP, SAA).
JNCI | Editorial 2 of 2
Downloaded from http://jnci.oxfordjournals.org/ at Univ of Iowa-Law Library on May 23, 2015
1. Olopade OI, Grushko TA, Nanda R, Huo D. Advances in breast cancer: pathways to personalized medicine. Clin Cancer Res. 2008;14(24):7988–7999. 2. US Breast Cancer Statistics. http://breastcancer.org. Accessed March 27, 2014. 3. Boyer B, Valles AM, Edme N. Induction and regulation of epithelial-mesenchymal transitions. Biochem Pharmacol. 2000;60(8):1091–1099. 4. Lee JM, Dedhar S, Kalluri R, Thompson EW. The epithelial-mesenchymal transition: new insights in signaling, development, and disease. J Cell Biol. 2006;172(7):973–981. 5. Kalluri R, Weinberg RA. The basics of epithelial-mesenchymal transition. J Clin Invest. 2009;119(6):1420–1428. 6. Zeisberg M, Neilson EG. Biomarkers for epithelial-mesenchymal transitions. J Clin Invest. 2009;119(6):1429–1437. 7. Kalluri R. EMT: when epithelial cells decide to become mesenchymal-like cells. J Clin Invest. 2009;119(6):1417–1419. 8. Thiery JP, Acloque H, Huang RY, Nieto MA. Epithelial-mesenchymal transitions in development and disease. Cell. 2009;139(5):871–890. 9. Gupta GP, Massague J. Cancer metastasis: building a framework. Cell. 2006;127(4):679–695. 10. Sleeman JP, Thiery JP. SnapShot: The epithelial-mesenchymal transition. Cell. 2011;145(1):162 e161. 11. Dobrovolskaia-Zavadskaia N. Sur la mortification spontanee de la queue chez la souris nouveau-nee et sur l’existence d’un caractere heriditaire “non-viable.” C R Soc Biol. 1927;97:114–116. 12. Wilson V, Conlon FL. The T-box family. Genome Biol. 2002;3(6):REVI EWS3008. 13. Atreya I, Schimanski CC, Becker C, et al. The T-box transcription factor eomesodermin controls CD8 T cell activity and lymph node metastasis in human colorectal cancer. Gut. 2007;56(11):1572–1578. 14. Arnold SJ, Hofmann UK, Bikoff EK, Robertson EJ. Pivotal roles for eomesodermin during axis formation, epithelium-to-mesenchyme transition and endoderm specification in the mouse. Development. 2008;135(3):501–511. 15. Takeichi M, Nimura K, Mori M, Nakagami H, Kaneda Y. The transcription factors Tbx18 and Wt1 control the epicardial epithelial-mesenchymal transition through bi-directional regulation of Slug in murine primary epicardial cells. PloS One. 2013;8(2):e57829. 16. Cai X, Nomura-Kitabayashi A, Cai W, Yan J, Christoffels VM, Cai CL. Myocardial Tbx20 regulates early atrioventricular canal formation and endocardial epithelial-mesenchymal transition via Bmp2. Develop Biol. 2011;360(2):381–390. 17. Rowley M, Grothey E, Couch FJ. The role of Tbx2 and Tbx3 in mammary development and tumorigenesis. J Mammary Gland Biol Neoplasia. 2004;9(2):109–118.
18. Sinclair CS, Adem C, Naderi A, et al. TBX2 is preferentially amplified in BRCA1- and BRCA2-related breast tumors. Cancer Res. 2002;62(13):3587–3591. 19. Wang B, Lindley LE, Fernandez-Vega V, Rieger ME, Sims AH, Briegel KJ. The T box transcription factor TBX2 promotes epithelial-mesenchymal transition and invasion of normal and malignant breast epithelial cells. PloS One. 2012;7(7):e41355. 20. Jacobs JJ, Keblusek P, Robanus-Maandag E, et al. Senescence bypass screen identifies TBX2, which represses Cdkn2a (p19(ARF)) and is amplified in a subset of human breast cancers. Nat Genet. 2000;26(3):291–299. 21. Rodriguez M, Aladowicz E, Lanfrancone L, Goding CR. Tbx3 represses E-cadherin expression and enhances melanoma invasiveness. Cancer Res. 2008;68(19):7872–7881. 22. Burgucu D, Guney K, Sahinturk D, et al. Tbx3 represses PTEN and is over-expressed in head and neck squamous cell carcinoma. BMC Cancer. October 19, 2012;12:481. 23. Humtsoe JO, Koya E, Pham E, et al. Transcriptional profiling identifies upregulated genes following induction of epithelial-mesenchymal transition in squamous carcinoma cells. Exp Cell Res. 2012;318(4):379–390. 24. Fan W, Huang X, Chen C, Gray J, Huang T. TBX3 and its isoform TBX3+2a are functionally distinctive in inhibition of senescence and are overexpressed in a subset of breast cancer cell lines. Cancer Res. 2004;64(15):5132–5139. 25. Yarosh W, Barrientos T, Esmailpour T, et al. TBX3 is overexpressed in breast cancer and represses p14 ARF by interacting with histone deacetylases. Cancer Res. 2008;68(3):693–699. 26. Palena C, Roselli M, Litzinger MT, et al. Brachyury, an EMT driver, is overexpressed in breast carcinomas and associates with poor prognosis. J Natl Cancer Inst. 2014;106(5): dju054 doi:10.1093/jnci/dju054. 27. Cheng Q, Chang JT, Geradts J, et al. Amplification and high-level expression of heat shock protein 90 marks aggressive phenotypes of human epidermal growth factor receptor 2 negative breast cancer. Breast Cancer Res. 2012;4(2):R62. 28. Matos LL, Trufelli DC, de Matos MG, da Silva Pinhal MA. Immunohistochemistry as an important tool in biomarkers detection and clinical practice. Biomarker Insights. February 9, 2010;5:9–20. 29. Taylor CR, Levenson RM. Quantification of immunohistochemistry-issues concerning methods, utility and semiquantitative assessment II. Histopathology. 2006;49(4):411–424. 30. Palena C, Fernando RI, Hamilton DH. An immunotherapeutic intervention against tumor progression: Targeting a driver of the epithelial-tomesenchymal transition. Oncoimmunology. 2014;3(1):e27220. 31. Hamilton DH, Litzinger MT, Fernando RI, Huang B, Palena C. Cancer vaccines targeting the epithelial-mesenchymal transition: tissue distribution of brachyury and other drivers of the mesenchymal-like phenotype of carcinomas. Semin Oncol. 2012;39(3):358–366. 32. Hamilton DH, Litzinger MT, Jales A, et al. Immunological targeting of tumor cells undergoing an epithelial-mesenchymal transition via a recombinant brachyury-yeast vaccine. Oncotarget. 2013;4(10):1777–1790. 33. Palena C, Polev DE, Tsang KY, et al. The human T-box mesodermal transcription factor Brachyury is a candidate target for T-cell-mediated cancer immunotherapy. Clin Cancer Res. 2007;13(8):2471–2478. 34. ClinicalTrials.gov. Open Label Study to Evaluate the Safety and Tolerability of GI-6301 a Vaccine Consisting of Whole Heat-Killed Recombinant Yeast Genetically Modified to Express Brachyury Protein in Adults with Solid Tumors. http://www.clinicaltrials.gov/ct2/show/results/
© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: [email protected].
Editorial
Brachyury: A New Player in Promoting Breast Cancer Aggressiveness Maira M. Pires, Stuart A. Aaronson Correspondence to: Stuart A. Aaronson, MD, Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Pl, Box 1130, New York, NY 10029 (e-mail: [email protected]).
1 of 2 Editorial | JNCI
cells to invade the extracellular matrix, 2) increased primary and secondary mammosphere formation, and 3) increased resistance to chemotherapy. The authors also sought to determine whether markers of stemness were altered by brachyury. Indeed, SOX2, NANOG and OCT4 were directly modulated by overexpression or downregulation of brachyury. Thus, Palena et al. (26) show for the first time that a member of the T-box TF family of proteins modulates these important pluripotency TFs. In comparing brachyury expression in normal and BC samples, Palena et al. (26) found that, whereas 21% of the 118 breast carcinoma samples analyzed were positive for brachyury mRNA expression by quantitative real-time polymerase chain reaction, approximately 90% of a different set of 30 primary breast tumors were positive by immunohistochemistry. Among 16 benign breast tissues examined, only two samples, both fibroadenomas, were positive by immunohistochemistry for this T-box protein. Further studies comparing the sensitivity of these approaches on the same samples should clarify the basis for the differences observed. For example, immunohistochemistry, which has been historically more difficult to quantitate (28,29), may overestimate the frequency of brachyury detection in BCs. Nonetheless, Palena et al.’s evidence associating brachyury with in vitro BC invasiveness, upregulation of stem cell TFs, and higher recurrence and metastatic spread suggests that brachyury could provide a new marker for aggressive breast tumors (26). Palena et al. (26) also developed a vector-based vaccine expressing brachyury aimed at targeting and killing brachyury-positive breast carcinoma cells to evaluate the possibility of a therapeutic application in BC patients. In fact, this vaccine, which uses a 9-mer peptide of brachyury that binds to human leukocyte antigen-A2, was shown previously to have efficacy in targeting other brachyury-positive tumor cells in vitro, such as lung cancer cells (30–33). Because their results demonstrated evidence of killing brachyurypositive BC cell lines in vitro, the authors argue that a brachyury vaccine approach could be considered as either mono- or combined therapy for the subset of BCs expressing this protein (26). In fact, a proposed brachyury-targeted vaccine is now being evaluated in a phase I clinical trial designed to establish safety and tolerability (34). Some questions raised by the work of Palena et al. also warrant further investigation. Although there was an indication that brachyury mRNA levels were higher in BCs negative for estrogen and progesterone receptors, brachyury was not correlated with a specific subtype of BC. Further studies encompassing larger numbers of BC samples will undoubtedly address whether brachyury Vol. 106, Issue 5 | dju094 | May 14, 2014
Downloaded from http://jnci.oxfordjournals.org/ at Univ of Iowa-Law Library on May 23, 2015
In the United States, breast cancer (BC) in women is the second most frequent cause of death and the second most diagnosed cancer, leading to approximately 230 000 new cases of invasive BC and approximately 40 000 new deaths a year (1,2). A major unmet need for all BCs is better understanding of what drives invasiveness and metastasis, factors responsible for the lethality of this disease. Epithelial-to-mesenchymal transition (EMT), initially described as a critical process in embryogenesis (3) and wound healing (4–7), has been recognized as a possible trigger for metastatic progression of tumors (4,8,9). This important and sometimes reversible process is associated with loss of cell-to-cell adhesion, increased cell motility and invasive properties, and specific morphogenetic changes (8,10). During EMT, certain epithelial markers, such as E-cadherin, are decreased or lost, whereas mesenchymal markers, such as N-cadherin, vimentin, snail, slug, and twist, are upregulated (4,8,10). The latter three are transcription factors (TFs) that are known to control EMT events associated with cancer progression. Brachyury, first discovered in 1927 (11), is the founding member of the highly conserved T-box TF family, whose members share a common DNA-binding domain of 180 to 200 amino acids known as the T-domain (12). Varying degrees of homology between the T-domain of T-box genes subdivide the family into subfamilies: Brachyury, T-brain I, Tbx1, Tbx2, and Tbx6. Within each subfamily, there may be multiple T-box genes (12). Over the past few years, several members of the T-box subfamilies, including brachyury, have been implicated in EMT and cancer. For example, in the T-brain I subfamily, eomesodermin has been reported to be not only involved in developmental EMT (13) but also in lymph node metastasis (14). Within the Tbx1 subfamily, both tbx18 and tbx20 can control different aspects of developmental EMT (15,16). In the Tbx2 subfamily, both tbx2 and tbx3 have been implicated in driving EMT and being overexpressed in cancers such as BCs (17–25). In this issue of the Journal, a new study by Palena et al. (26) focuses on the role of brachyury in BC. This study classified more than 4000 breast tumor samples for their levels of brachyury mRNA expression from 23 published databases from the Gene Expression Omnibus (27). After brachyury levels were subgrouped into low, intermediate, and high expression, Kaplan–Meier analysis was performed on a set of 357 BC patients treated with adjuvant tamoxifen monotherapy for 5 years to assess prognostic differences among the three subgroups. High brachyury mRNA was associated with higher risk of recurrence and distant metastasis. Furthermore, exogenous modulation of brachyury expression in BC cells revealed that higher brachyury levels 1) enhanced the ability of such BC
overexpression is more specific to a particular subtype of BC or whether it is a general marker for BC metastasis and poor survival. Furthermore, mechanistic dissection of brachyury downstream target genes responsible for mediating its invasive effects could prove valuable in identifying future therapeutic targets. Brachyury is now the third member of the T-box proteins, along with tbx2 and tbx3, to be implicated in driving various aspects of BC biology, such as induction of EMT and invasiveness. Palena et al.’s findings (26) contribute to our knowledge of a new driver of the metastatic phenotype of BC cells and suggest that future investigations of other less well-characterized T-box protein family members should provide a more complete picture of the importance of T-box TFs in driving BC and other carcinomas. References
jnci.oxfordjournals.org
NCT01519817. Accessed March 27, 2014.
Notes The authors have no conflicts of interest to declare. Affiliation of authors: Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY (MMP, SAA).
JNCI | Editorial 2 of 2
Downloaded from http://jnci.oxfordjournals.org/ at Univ of Iowa-Law Library on May 23, 2015
1. Olopade OI, Grushko TA, Nanda R, Huo D. Advances in breast cancer: pathways to personalized medicine. Clin Cancer Res. 2008;14(24):7988–7999. 2. US Breast Cancer Statistics. http://breastcancer.org. Accessed March 27, 2014. 3. Boyer B, Valles AM, Edme N. Induction and regulation of epithelial-mesenchymal transitions. Biochem Pharmacol. 2000;60(8):1091–1099. 4. Lee JM, Dedhar S, Kalluri R, Thompson EW. The epithelial-mesenchymal transition: new insights in signaling, development, and disease. J Cell Biol. 2006;172(7):973–981. 5. Kalluri R, Weinberg RA. The basics of epithelial-mesenchymal transition. J Clin Invest. 2009;119(6):1420–1428. 6. Zeisberg M, Neilson EG. Biomarkers for epithelial-mesenchymal transitions. J Clin Invest. 2009;119(6):1429–1437. 7. Kalluri R. EMT: when epithelial cells decide to become mesenchymal-like cells. J Clin Invest. 2009;119(6):1417–1419. 8. Thiery JP, Acloque H, Huang RY, Nieto MA. Epithelial-mesenchymal transitions in development and disease. Cell. 2009;139(5):871–890. 9. Gupta GP, Massague J. Cancer metastasis: building a framework. Cell. 2006;127(4):679–695. 10. Sleeman JP, Thiery JP. SnapShot: The epithelial-mesenchymal transition. Cell. 2011;145(1):162 e161. 11. Dobrovolskaia-Zavadskaia N. Sur la mortification spontanee de la queue chez la souris nouveau-nee et sur l’existence d’un caractere heriditaire “non-viable.” C R Soc Biol. 1927;97:114–116. 12. Wilson V, Conlon FL. The T-box family. Genome Biol. 2002;3(6):REVI EWS3008. 13. Atreya I, Schimanski CC, Becker C, et al. The T-box transcription factor eomesodermin controls CD8 T cell activity and lymph node metastasis in human colorectal cancer. Gut. 2007;56(11):1572–1578. 14. Arnold SJ, Hofmann UK, Bikoff EK, Robertson EJ. Pivotal roles for eomesodermin during axis formation, epithelium-to-mesenchyme transition and endoderm specification in the mouse. Development. 2008;135(3):501–511. 15. Takeichi M, Nimura K, Mori M, Nakagami H, Kaneda Y. The transcription factors Tbx18 and Wt1 control the epicardial epithelial-mesenchymal transition through bi-directional regulation of Slug in murine primary epicardial cells. PloS One. 2013;8(2):e57829. 16. Cai X, Nomura-Kitabayashi A, Cai W, Yan J, Christoffels VM, Cai CL. Myocardial Tbx20 regulates early atrioventricular canal formation and endocardial epithelial-mesenchymal transition via Bmp2. Develop Biol. 2011;360(2):381–390. 17. Rowley M, Grothey E, Couch FJ. The role of Tbx2 and Tbx3 in mammary development and tumorigenesis. J Mammary Gland Biol Neoplasia. 2004;9(2):109–118.
18. Sinclair CS, Adem C, Naderi A, et al. TBX2 is preferentially amplified in BRCA1- and BRCA2-related breast tumors. Cancer Res. 2002;62(13):3587–3591. 19. Wang B, Lindley LE, Fernandez-Vega V, Rieger ME, Sims AH, Briegel KJ. The T box transcription factor TBX2 promotes epithelial-mesenchymal transition and invasion of normal and malignant breast epithelial cells. PloS One. 2012;7(7):e41355. 20. Jacobs JJ, Keblusek P, Robanus-Maandag E, et al. Senescence bypass screen identifies TBX2, which represses Cdkn2a (p19(ARF)) and is amplified in a subset of human breast cancers. Nat Genet. 2000;26(3):291–299. 21. Rodriguez M, Aladowicz E, Lanfrancone L, Goding CR. Tbx3 represses E-cadherin expression and enhances melanoma invasiveness. Cancer Res. 2008;68(19):7872–7881. 22. Burgucu D, Guney K, Sahinturk D, et al. Tbx3 represses PTEN and is over-expressed in head and neck squamous cell carcinoma. BMC Cancer. October 19, 2012;12:481. 23. Humtsoe JO, Koya E, Pham E, et al. Transcriptional profiling identifies upregulated genes following induction of epithelial-mesenchymal transition in squamous carcinoma cells. Exp Cell Res. 2012;318(4):379–390. 24. Fan W, Huang X, Chen C, Gray J, Huang T. TBX3 and its isoform TBX3+2a are functionally distinctive in inhibition of senescence and are overexpressed in a subset of breast cancer cell lines. Cancer Res. 2004;64(15):5132–5139. 25. Yarosh W, Barrientos T, Esmailpour T, et al. TBX3 is overexpressed in breast cancer and represses p14 ARF by interacting with histone deacetylases. Cancer Res. 2008;68(3):693–699. 26. Palena C, Roselli M, Litzinger MT, et al. Brachyury, an EMT driver, is overexpressed in breast carcinomas and associates with poor prognosis. J Natl Cancer Inst. 2014;106(5): dju054 doi:10.1093/jnci/dju054. 27. Cheng Q, Chang JT, Geradts J, et al. Amplification and high-level expression of heat shock protein 90 marks aggressive phenotypes of human epidermal growth factor receptor 2 negative breast cancer. Breast Cancer Res. 2012;4(2):R62. 28. Matos LL, Trufelli DC, de Matos MG, da Silva Pinhal MA. Immunohistochemistry as an important tool in biomarkers detection and clinical practice. Biomarker Insights. February 9, 2010;5:9–20. 29. Taylor CR, Levenson RM. Quantification of immunohistochemistry-issues concerning methods, utility and semiquantitative assessment II. Histopathology. 2006;49(4):411–424. 30. Palena C, Fernando RI, Hamilton DH. An immunotherapeutic intervention against tumor progression: Targeting a driver of the epithelial-tomesenchymal transition. Oncoimmunology. 2014;3(1):e27220. 31. Hamilton DH, Litzinger MT, Fernando RI, Huang B, Palena C. Cancer vaccines targeting the epithelial-mesenchymal transition: tissue distribution of brachyury and other drivers of the mesenchymal-like phenotype of carcinomas. Semin Oncol. 2012;39(3):358–366. 32. Hamilton DH, Litzinger MT, Jales A, et al. Immunological targeting of tumor cells undergoing an epithelial-mesenchymal transition via a recombinant brachyury-yeast vaccine. Oncotarget. 2013;4(10):1777–1790. 33. Palena C, Polev DE, Tsang KY, et al. The human T-box mesodermal transcription factor Brachyury is a candidate target for T-cell-mediated cancer immunotherapy. Clin Cancer Res. 2007;13(8):2471–2478. 34. ClinicalTrials.gov. Open Label Study to Evaluate the Safety and Tolerability of GI-6301 a Vaccine Consisting of Whole Heat-Killed Recombinant Yeast Genetically Modified to Express Brachyury Protein in Adults with Solid Tumors. http://www.clinicaltrials.gov/ct2/show/results/
