Open Access Highly Accessed Research article

PAM50 Breast Cancer Subtyping by RT-qPCR and Concordance with Standard Clinical Molecular Markers

Roy RL Bastien1, Álvaro Rodríguez-Lescure2, Mark TW Ebbert1, Aleix Prat34, Blanca Munárriz5, Leslie Rowe1, Patricia Miller1, Manuel Ruiz-Borrego6, Daniel Anderson1, Bradley Lyons1, Isabel Álvarez7, Tracy Dowell1, David Wall1, Miguel Ángel Seguí8, Lee Barley1, Kenneth M Boucher9, Emilio Alba10, Lisa Pappas9, Carole A Davis11, Ignacio Aranda12, Christiane Fauron1, Inge J Stijleman11, José Palacios13, Antonio Antón14, Eva Carrasco15, Rosalía Caballero15, Matthew J Ellis16, Torsten O Nielsen17, Charles M Perou3, Mark Astill1, Philip S Bernard11119* and Miguel Martín18

Author affiliations

1 The ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, USA

2 Department of Medical Oncology, Hospital Universitario de Elche, Elche, Spain

3 Lineberger Comprehensive Cancer Center and Department of Genetics and Department of Pathology & Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

4 Department of Medicine, Universitat Autónoma de Barcelona, Barcelona, Spain

5 Department of Medical Oncology, Hospital Universitario La Fe, Valencia, Spain

6 Department of Medical Oncology, Hospital Universitario Virgen del Rocío, Sevilla, Spain

7 Department of Medical Oncology, Hospital de Donostia, San Sebastián, Spain

8 Department of Medical Oncology, Corporatiò Sanitaria Parc Taulí, Sabadell, Spain

9 Department of Oncological Sciences, Huntsman Cancer Institute, Salt Lake City, UT, USA

10 Department of Medical Oncology, Hospital Universitario Virgen de la Victoria, Málaga, Spain

11 Department of Pathology, University of Utah Health Sciences Center/Huntsman Cancer Institute, Salt Lake City, UT, USA

12 Department of Pathology, Hospital General Universitario de Alicante, Alicante, Spain

13 Department of Pathology, Hospital Virgen del Rocio, Sevilla, Spain

14 Department of Medical Oncology, Hospital Universitario Miguel Servet, Zaragoza, Spain

15 Spanish Breast Cancer Research Group, GEICAM, Madrid, Spain

16 Department of Oncology, Washington University, St. Louis, MO, USA

17 Department of Anatomical Pathology, University of British Columbia, Vancouver, Canada

18 Department of Medical Oncology, Hospital General Universitario Gregorio Marañón, Universidad Complutense, Madrid, Spain

19 Huntsman Cancer Institute, 2000 Circle of Hope, Salt Lake City, UT, 84112, USA

For all author emails, please log on.

Citation and License

BMC Medical Genomics 2012, 5:44  doi:10.1186/1755-8794-5-44

Published: 4 October 2012



Many methodologies have been used in research to identify the “intrinsic” subtypes of breast cancer commonly known as Luminal A, Luminal B, HER2-Enriched (HER2-E) and Basal-like. The PAM50 gene set is often used for gene expression-based subtyping; however, surrogate subtyping using panels of immunohistochemical (IHC) markers are still widely used clinically. Discrepancies between these methods may lead to different treatment decisions.


We used the PAM50 RT-qPCR assay to expression profile 814 tumors from the GEICAM/9906 phase III clinical trial that enrolled women with locally advanced primary invasive breast cancer. All samples were scored at a single site by IHC for estrogen receptor (ER), progesterone receptor (PR), and Her2/neu (HER2) protein expression. Equivocal HER2 cases were confirmed by chromogenic in situ hybridization (CISH). Single gene scores by IHC/CISH were compared with RT-qPCR continuous gene expression values and “intrinsic” subtype assignment by the PAM50. High, medium, and low expression for ESR1, PGR, ERBB2, and proliferation were selected using quartile cut-points from the continuous RT-qPCR data across the PAM50 subtype assignments.


ESR1, PGR, and ERBB2 gene expression had high agreement with established binary IHC cut-points (area under the curve (AUC) ≥ 0.9). Estrogen receptor positivity by IHC was strongly associated with Luminal (A and B) subtypes (92%), but only 75% of ER negative tumors were classified into the HER2-E and Basal-like subtypes. Luminal A tumors more frequently expressed PR than Luminal B (94% vs 74%) and Luminal A tumors were less likely to have high proliferation (11% vs 77%). Seventy-seven percent (30/39) of ER-/HER2+ tumors by IHC were classified as the HER2-E subtype. Triple negative tumors were mainly comprised of Basal-like (57%) and HER2-E (30%) subtypes. Single gene scoring for ESR1, PGR, and ERBB2 was more prognostic than the corresponding IHC markers as shown in a multivariate analysis.


The standard immunohistochemical panel for breast cancer (ER, PR, and HER2) does not adequately identify the PAM50 gene expression subtypes. Although there is high agreement between biomarker scoring by protein immunohistochemistry and gene expression, the gene expression determinations for ESR1 and ERBB2 status was more prognostic.