Triple-negative breast cancer (TNBC) has a worse prognosis compared with other breast cancer subtypes, and biomarkers to identify patients at high risk of recurrence are needed. Here, we investigated the expression of human epidermal receptor (HER) family members in TNBC and evaluated their potential as biomarkers of recurrence.
We developed Time Resolved-Förster Resonance Energy Transfer (TR-FRET) assays to quantify HER1, HER2 and HER3 in formalin-fixed paraffin-embedded (FFPE) tumour tissues. After assessing the performance and precision of our assays, we quantified HER protein expression in 51 TNBC specimens, and investigated the association of their expression with relapse-free survival.
The assays were quantitative, accurate, and robust. In TNBC specimens, HER1 levels ranged from ≈4000 to more than 2 million receptors per cell, whereas HER2 levels varied from ≈1000 to 60,000 receptors per cell. HER3 expression was very low (less than 5500 receptors per cell in all samples). Moderate HER2 expression was significantly associated with higher risk of recurrence (HR = 3.93; P = 0.003).
Our TR-FRET assays accurately quantify HER1, HER2 and HER3 in FFPE breast tumour specimens. Moderate HER2 expression may represent a novel prognostic marker in patients with TNBC.
Subscribe to Journal
Get full journal access for 1 year
only $32.88 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Brenton, J. D., Carey, L. A., Ahmed, A. A. & Caldas, C. Molecular classification and molecular forecasting of breast cancer: ready for clinical application? J. Clin. Oncol. 23, 7350–7360 (2005).
Papadimitriou, M., Mountzios, G. & Papadimitriou, C. A. The role of PARP inhibition in triple-negative breast cancer: Unraveling the wide spectrum of synthetic lethality. Cancer Treat. Rev. 67, 34–44 (2018).
Dent, R., Trudeau, M., Pritchard, K. I., Hanna, W. M., Kahn, H. K., Sawka, C. A. et al. Triple-negative breast cancer: clinical features and patterns of recurrence. Clin. Cancer 13, 4429–4434 (2007).
Bauer, K. R., Brown, M., Cress, R. D., Parise, C. A. & Caggiano, V. Descriptive analysis of estrogen receptor (ER)-negative, progesterone receptor (PR)-negative, and HER2-negative invasive breast cancer, the so-called triple-negative phenotype: a population-based study from the California cancer Registry. Cancer 109, 1721–1728 (2007).
Bianchini, G., Balko, J. M., Mayer, I. A., Sanders, M. E. & Gianni, L. Triple-negative breast cancer: challenges and opportunities of a heterogeneous disease. Nat. Rev. Clin. Oncol. 13, 674–690 (2016).
Lehmann, B. D., Bauer, J. A., Chen, X., Sanders, M. E., Chakravarthy, A. B., Shyr, Y. et al. Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies. J. Clin. Invest. 121, 2750–2767 (2011).
Burstein, M. D., Tsimelzon, A., Poage, G. M., Covington, K. R., Contreras, A., Fuqua, S. A. W. et al. Comprehensive genomic analysis identifies novel subtypes and targets of triple-negative breast cancer. Clin. Cancer Res. 21, 1688–1698 (2015).
Masuda, H., Baggerly, K. A., Wang, Y., Zhang, Y., Gonzalez-Angulo, A. M., Meric-Bernstam, F. et al. Differential response to neoadjuvant chemotherapy among 7 triple-negative breast cancer molecular subtypes. Clin. Cancer Res. 19, 5533–5540 (2013).
Yarden, Y. & Sliwkowski, M. X. Untangling the ErbB signalling network. Nat. Rev. Mol. Cell Biol. 2, 127–137 (2001).
Gonzalez-Conchas, G. A., Rodriguez-Romo, L., Hernandez-Barajas, D., Gonzalez-Guerrero, J. F., Rodriguez-Fernandez, I. A., Verdines-Perez, A. et al. Epidermal growth factor receptor overexpression and outcomes in early breast cancer: A systematic review and a meta-analysis. Cancer Treat. Rev. 62, 1–8 (2017).
Hoadley, K. A., Weigman, V. J., Fan, C., Sawyer, L. R., He, X., Troester, M. A. et al. EGFR associated expression profiles vary with breast tumor subtype. BMC Genomics 8, 258 (2007).
Costa, R., Shah, A. N., Santa-Maria, C. A., Cruz, M. R., Mahalingam, D., Carneiro, B. A. et al. Targeting epidermal growth factor receptor in triple negative breast cancer: new discoveries and practical insights for drug development. Cancer Treat. Rev. 53, 111–119 (2017).
Paik, S., Kim, C. & Wolmark, N. HER2 status and benefit from adjuvant trastuzumab in breast cancer. N. Engl. J. Med. 358, 1409–1411 (2008).
Perez, E. A., Reinholz, M. M., Hillman, D. W., Tenner, K. S., Schroeder, M. J., Davidson, N. E. et al. HER2 and chromosome 17 effect on patient outcome in the N9831 adjuvant trastuzumab trial. J. Clin. Oncol. 28, 4307–4315 (2010).
Fehrenbacher, L., Jeong, J.-H., Rastogi, P., Geyer, C. E., Paik, S., Ganz, P. A. et al. OT1-02-07: NSABP B-47: A Randomized Phase III Trial of Adjuvant Therapy Comparing Chemotherapy Alone (Six Cycles of Docetaxel Plus Cyclophosphamide or Four Cycles of Doxorubicin Plus Cyclophosphamide Followed by Weekly Paclitaxel) to Chemotherapy Plus Trastuzumab in Women with Node-Positive or High-Risk Node-Negative HER2−Low Invasive Breast Cancer. Cancer Res. 71(Suppl 24), OT1-02–OT1-007 (2011).
Fehrenbacher, L., Cecchini, R. S., Geyer, C. E., Rastogi, P., Costantino, J. P., Atkins, J. N. et al. Abstract GS1-02: NSABP B-47 (NRG oncology): Phase III randomized trial comparing adjuvant chemotherapy with adriamycin (A) and cyclophosphamide (C) → weekly paclitaxel (WP), or docetaxel (T) and C with or without a year of trastuzumab (H) in women with node-positive or high-risk node-negative invasive breast cancer (IBC) expressing HER2 staining intensity of IHC 1+ or 2+ with negative FISH (HER2-Low IBC). Cancer Res. 78(Suppl 4), GS1–GS02 (2018).
Ilie, S. M., Desauw, C. & Hebbar, M. HER2 based expression subpopulations in TNBC: pathological aspects and clinical significance. Ann. Oncol. 27(Suppl 6), vi15–vi42 (2016).
Bae, S. Y., La Choi, Y., Kim, S., Kim, M., Kim, J., Jung, S. P. et al. HER3 status by immunohistochemistry is correlated with poor prognosis in hormone receptor-negative breast cancer patients. Breast Cancer Res. Treat. 139, 741–750 (2013).
Goldstein, N. S., Hewitt, S. M., Taylor, C. R., Yaziji, H. & Hicks, D. G., Members of Ad-Hoc Committee On Immunohistochemistry Standardization. Recommendations for improved standardization of immunohistochemistry. Appl. Immunohistochem. Mol. Morphol. 15, 124–133 (2007).
Perez, E. A., Press, M. F., Dueck, A. C., Jenkins, R. B., Kim, C., Chen, B. et al. Immunohistochemistry and fluorescence in situ hybridization assessment of HER2 in clinical trials of adjuvant therapy for breast cancer (NCCTG N9831, BCIRG 006, and BCIRG 005). Breast Cancer Res. Treat. 138, 99–108 (2013).
McCullough, A. E., Dell’orto, P., Reinholz, M. M., Gelber, R. D., Dueck, A. C., Russo, L. et al. Central pathology laboratory review of HER2 and ER in early breast cancer: an ALTTO trial [BIG 2-06/NCCTG N063D (Alliance)] ring study. Breast Cancer Res. Treat. 143, 485–492 (2014).
Mathis, G. HTRF(R) technology. J. Biomol. Screen 4, 309–314 (1999).
Ho-Pun-Cheung, A., Bazin, H., Gaborit, N., Larbouret, C., Garnero, P., Assenat, E. et al. Quantification of HER expression and dimerization in patients’ tumor samples using time-resolved Förster resonance energy transfer. PloS ONE 7, e37065 (2012).
Lopez-Crapez, E., Ho-Pun-Cheung, A., Garnero, P. & Bazin, H. Evaluation of the dimerization profiles of HER tyrosine kinases by time-resolved Förster resonance energy transfer (TR-FRET). Methods Mol. Biol. Clifton NJ 1233, 45–55 (2015).
Wolff, A. C., Hammond, M. E. H., Schwartz, J. N., Hagerty, K. L., Allred, D. C., Cote, R. J. et al. 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, 18–43 (2007).
Albizu, L., Cottet, M., Kralikova, M., Stoev, S., Seyer, R., Brabet, I. et al. Time-resolved FRET between GPCR ligands reveals oligomers in native tissues. Nat. Chem. Biol. 6, 587–594 (2010).
McCarty, K. S., Szabo, E., Flowers, J. L., Cox, E. B., Leight, G. S., Miller, L. et al. Use of a monoclonal anti-estrogen receptor antibody in the immunohistochemical evaluation of human tumors. Cancer Res. 46(Suppl 8), 4244s–4248s (1986).
Silva, L. P., Lorenzi, P. L., Purwaha, P., Yong, V., Hawke, D. H. & Weinstein, J. N. Measurement of DNA concentration as a normalization strategy for metabolomic data from adherent cell lines. Anal. Chem. 85, 9536–9542 (2013).
Lewin, B. Genes V 5th edn. (Oxford University Press, Oxford, England, 1994).
DeSouza, L. V., Krakovska, O., Darfler, M. M., Krizman, D. B., Romaschin, A. D., Colgan, T. J. et al. mTRAQ-based quantification of potential endometrial carcinoma biomarkers from archived formalin-fixed paraffin-embedded tissues. Proteomics 10, 3108–3116 (2010).
Larson, J. S., Goodman, L. J., Tan, Y., Defazio-Eli, L., Paquet, A. C., Cook, J. W. et al. Analytical Validation of a Highly Quantitative, Sensitive, Accurate, and Reproducible Assay (HERmark) for the Measurement of HER2 Total Protein and HER2 Homodimers in FFPE Breast Cancer Tumor Specimens. Pathol. Res. Int. 2010, 814176 (2010).
Rakha, E. A., El-Sayed, M. E., Green, A. R., Lee, A. H. S., Robertson, J. F. & Ellis, I. O. Prognostic markers in triple-negative breast cancer. Cancer 109, 25–32 (2007).
Nakai, K., Hung, M.-C. & Yamaguchi, H. A perspective on anti-EGFR therapies targeting triple-negative breast cancer. Am. J. Cancer Res. 6, 1609–1623 (2016).
Zhang, Q., Park, E., Kani, K. & Landgraf, R. Functional isolation of activated and unilaterally phosphorylated heterodimers of ERBB2 and ERBB3 as scaffolds in ligand-dependent signaling. Proc. Natl Acad. Sci. USA 109, 13237–13242 (2012).
Capuani, F., Conte, A., Argenzio, E., Marchetti, L., Priami, C., Polo, S. et al. Quantitative analysis reveals how EGFR activation and downregulation are coupled in normal but not in cancer cells. Nat. Commun. 6, 7999 (2015).
Witton, C. J., Reeves, J. R., Going, J. J., Cooke, T. G. & Bartlett, J. M. S. Expression of the HER1-4 family of receptor tyrosine kinases in breast cancer. J. Pathol. 200, 290–297 (2003).
Tanner, B., Hasenclever, D., Stern, K., Schormann, W., Bezler, M., Hermes, M. et al. ErbB-3 predicts survival in ovarian cancer. J. Clin. Oncol. 24, 4317–4323 (2006).
Ithimakin, S., Day, K. C., Malik, F., Zen, Q., Dawsey, S. J., Bersano-Begey, T. F. et al. HER2 drives luminal breast cancer stem cells in the absence of HER2 amplification: implications for efficacy of adjuvant trastuzumab. Cancer Res. 73, 1635–1646 (2013).
Phillips, T. M., McBride, W. H. & Pajonk, F. The response of CD24(-/low)/CD44+ breast cancer-initiating cells to radiation. J. Natl Cancer Inst. 98, 1777–1785 (2006).
Shafee, N., Smith, C. R., Wei, S., Kim, Y., Mills, G. B., Hortobagyi, G. N. et al. Cancer stem cells contribute to cisplatin resistance in Brca1/p53-mediated mouse mammary tumors. Cancer Res. 68, 3243–3250 (2008).
Li, X., Lewis, M. T., Huang, J., Gutierrez, C., Osborne, C. K., Wu, M.-F. et al. Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy. J. Natl Cancer Inst. 100, 672–679 (2008).
O’Hurley, G., Sjöstedt, E., Rahman, A., Li, B., Kampf, C., Pontén, F. et al. Garbage in, garbage out: a critical evaluation of strategies used for validation of immunohistochemical biomarkers. Mol. Oncol. 8, 783–798 (2014).
Steiner, C., Tille, J.-C., Lamerz, J., Kux van Geijtenbeek, S., McKee, T. A., Venturi, M. et al. Quantification of HER2 by targeted mass spectrometry in formalin-fixed paraffin-embedded (FFPE) breast cancer tissues. Mol. Cell Proteomics 14, 2786–2799 (2015).
Shi, Y., Huang, W., Tan, Y., Jin, X., Dua, R., Penuel, E. et al. A novel proximity assay for the detection of proteins and protein complexes: quantitation of HER1 and HER2 total protein expression and homodimerization in formalin-fixed, paraffin-embedded cell lines and breast cancer tissue. Diagn. Mol. Pathol. 18, 11–21 (2009).
Desmedt, C., Sperinde, J., Piette, F., Huang, W., Jin, X., Tan, Y. et al. Quantitation of HER2 expression or HER2:HER2 dimers and differential survival in a cohort of metastatic breast cancer patients carefully selected for trastuzumab treatment primarily by FISH. Diagn. Mol. Pathol. 18, 22–29 (2009).
Nuciforo, P., Radosevic-Robin, N., Ng, T. & Scaltriti, M. Quantification of HER family receptors in breast cancer. Breast Cancer Res. 17, 53 (2015).
Sperinde, J., Huang, W., Vehtari, A., Chenna, A., Kellokumpu-Lehtinen, P. L., Winslow, J. et al. p95HER2 methionine 611 carboxy-terminal fragment is predictive of trastuzumab adjuvant treatment benefit in the FinHer trial. Clin. Cancer Res. 24, 3046–3052 (2018).
Chumsri, S., Sperinde, J., Liu, H., Gligorov, J., Spano, J. P., Antoine, M. et al. High p95HER2/HER2 ratio associated with poor outcome in trastuzumab-treated HER2-positive metastatic breast cancer NCCTG N0337 and NCCTG 98-32-52 (Alliance). Clin. Cancer Res. 24, 3053–3058 (2018).
We thank Magalie Pedot for her excellent technical assistance. We are grateful to Blandine Massemin for her help in collecting and preparing clinical samples. We also thank Sonia Boulabas for collecting the clinical data.
Ethical approval and consent to participate
The present study was performed in accordance with the Declaration of Helsinki guidelines, and was approved by the ICM Institutional Review Board (ID number ICM-CRB-2012/04). Samples were provided by ICM biological resource centre (Biobank number BB-0033-00059), following the ethics and legal national French dispositions for the patients’ information and consent. Considering the retrospective, non-interventional nature of this study, no consent was deemed necessary by the review board.
Consent for publication
Our manuscript does not contain any individual person’s data in any form.
The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.
The authors declare no competing interests.
This work was supported by the grant AAP13 “Fonds Unique Interministériel” FUI UmAbHER3 F120402M.
Note This work is published under the standard license to publish agreement. After 12 months the work will become freely available and the license terms will switch to a Creative Commons Attribution 4.0 International (CC BY 4.0).
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Ho-Pun-Cheung, A., Bazin, H., Boissière-Michot, F. et al. Quantification of HER1, HER2 and HER3 by time-resolved Förster resonance energy transfer in FFPE triple-negative breast cancer samples. Br J Cancer (2019) doi:10.1038/s41416-019-0670-8