title: PAM50 breast cancer subtyping by RT-qPCR and concordance with standard clinical molecular markers.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: The ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, USA.
authors.forename: Roy R L
authors.initials: RR
authors.lastname: Bastien
authors.forename: Álvaro
authors.initials: Á
authors.lastname: Rodríguez-Lescure
authors.forename: Mark T W
authors.initials: MT
authors.lastname: Ebbert
authors.forename: Aleix
authors.initials: A
authors.lastname: Prat
authors.forename: Blanca
authors.initials: B
authors.lastname: Munárriz
authors.forename: Leslie
authors.initials: L
authors.lastname: Rowe
authors.forename: Patricia
authors.initials: P
authors.lastname: Miller
authors.forename: Manuel
authors.initials: M
authors.lastname: Ruiz-Borrego
authors.forename: Daniel
authors.initials: D
authors.lastname: Anderson
authors.forename: Bradley
authors.initials: B
authors.lastname: Lyons
authors.forename: Isabel
authors.initials: I
authors.lastname: Álvarez
authors.forename: Tracy
authors.initials: T
authors.lastname: Dowell
authors.forename: David
authors.initials: D
authors.lastname: Wall
authors.forename: Miguel Ángel
authors.initials: MÁ
authors.lastname: Seguí
authors.forename: Lee
authors.initials: L
authors.lastname: Barley
authors.forename: Kenneth M
authors.initials: KM
authors.lastname: Boucher
authors.forename: Emilio
authors.initials: E
authors.lastname: Alba
authors.forename: Lisa
authors.initials: L
authors.lastname: Pappas
authors.forename: Carole A
authors.initials: CA
authors.lastname: Davis
authors.forename: Ignacio
authors.initials: I
authors.lastname: Aranda
authors.forename: Christiane
authors.initials: C
authors.lastname: Fauron
authors.forename: Inge J
authors.initials: IJ
authors.lastname: Stijleman
authors.forename: José
authors.initials: J
authors.lastname: Palacios
authors.forename: Antonio
authors.initials: A
authors.lastname: Antón
authors.forename: Eva
authors.initials: E
authors.lastname: Carrasco
authors.forename: Rosalía
authors.initials: R
authors.lastname: Caballero
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Ellis
authors.forename: Torsten O
authors.initials: TO
authors.lastname: Nielsen
authors.forename: Charles M
authors.initials: CM
authors.lastname: Perou
authors.forename: Mark
authors.initials: M
authors.lastname: Astill
authors.forename: Philip S
authors.initials: PS
authors.lastname: Bernard
authors.forename: Miguel
authors.initials: M
authors.lastname: Martín
mesh_terms: D014408:Biomarkers, Tumor / Q000235:genetics* / Q000378:metabolism; D001940:Breast / Q000378:metabolism / Q000473:pathology; D001943:Breast Neoplasms / Q000145:classification* / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002986:Clinical Trials as Topic; D016000:Cluster Analysis; D047628:Estrogen Receptor alpha / Q000378:metabolism; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007150:Immunohistochemistry; D053208:Kaplan-Meier Estimate; D057230:Limit of Detection; D015999:Multivariate Analysis; D011379:Prognosis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012372:ROC Curve; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
chemical_list: D014408:Biomarkers, Tumor; D047628:Estrogen Receptor alpha; D012333:RNA, Messenger; D018719:Erb-b2 Receptor Tyrosine Kinases; C506487:ESR1 protein, human; C508053:ERBB2 protein, human