title: Comparison of immunohistochemistry with PCR for assessment of ER, PR, and Ki-67 and prediction of pathological complete response in breast cancer.
abstract: Proliferation may predict response to neoadjuvant therapy of breast cancer and is commonly assessed by manual scoring of slides stained by immunohistochemistry (IHC) for Ki-67 similar to ER and PgR. This method carries significant intra- and inter-observer variability. Automatic scoring of Ki-67 with digital image analysis (qIHC) or assessment of MKI67 gene expression with RT-qPCR may improve diagnostic accuracy.
  
  
  
  Ki-67 IHC visual assessment was compared to the IHC nuclear tool (AperioTM) on core biopsies from a randomized neoadjuvant clinical trial. Expression of ESR1, PGR and MKI67 by RT-qPCR was performed on RNA extracted from the same formalin-fixed paraffin-embedded tissue. Concordance between the three methods (vIHC, qIHC and RT-qPCR) was assessed for all 3 markers. The potential of Ki-67 IHC and RT-qPCR to predict pathological complete response (pCR) was evaluated using ROC analysis and non-parametric Mann-Whitney Test.
  
  
  
  Correlation between methods (qIHC versus RT-qPCR) was high for ER and PgR (spearman´s r = 0.82, p < 0.0001 and r = 0.86, p < 0.0001, respectively) resulting in high levels of concordance using predefined cut-offs. When comparing qIHC of ER and PgR with RT-qPCR of ESR1 and PGR the overall agreement was 96.6 and 91.4%, respectively, while overall agreement of visual IHC with RT-qPCR was slightly lower for ER/ESR1 and PR/PGR (91.2 and 92.9%, respectively). In contrast, only a moderate correlation was observed between qIHC and RT-qPCR continuous data for Ki-67/MKI67 (Spearman's r = 0.50, p = 0.0001). Up to now no predictive cut-off for Ki-67 assessment by IHC has been established to predict response to neoadjuvant chemotherapy. Setting the desired sensitivity at 100%, specificity for the prediction of pCR (ypT0ypN0) was significantly higher for mRNA than for protein (68.9% vs. 22.2%). Moreover, the proliferation levels in patients achieving a pCR versus not differed significantly using MKI67 RNA expression (Mann-Whitney p = 0.002), but not with qIHC of Ki-67 (Mann-Whitney p = 0.097) or vIHC of Ki-67 (p = 0.131).
  
  
  
  Digital image analysis can successfully be implemented for assessing ER, PR and Ki-67. IHC for ER and PR reveals high concordance with RT-qPCR. However, RT-qPCR displays a broader dynamic range and higher sensitivity than IHC. Moreover, correlation between Ki-67 qIHC and RT-qPCR is only moderate and RT-qPCR with MammaTyper® outperforms qIHC in predicting pCR. Both methods yield improvements to error-prone manual scoring of Ki-67. However, RT-qPCR was significantly more specific.
authors.affiliation: Institute of Pathology, University of Heidelberg, Im Neuenheimer Feld 220-221, 69120, Heidelberg, Germany. peter.sinn@med.uni-heidelberg.de.
authors.forename: Hans-Peter
authors.initials: HP
authors.lastname: Sinn
authors.affiliation: National Center for Tumor Diseases, University-Hospital Heidelberg, Im Neuenheimer Feld 460, 69120, Heidelberg, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Schneeweiss
authors.affiliation: Institute of Pathology, University of Heidelberg, Im Neuenheimer Feld 220-221, 69120, Heidelberg, Germany.
authors.forename: Marius
authors.initials: M
authors.lastname: Keller
authors.affiliation: BioNTech Diagnostics GmbH, 55131, Mainz, Germany.
authors.forename: Kornelia
authors.initials: K
authors.lastname: Schlombs
authors.affiliation: BioNTech Diagnostics GmbH, 55131, Mainz, Germany.
authors.forename: Mark
authors.initials: M
authors.lastname: Laible
authors.affiliation: National Center for Tumor Diseases, University-Hospital Heidelberg, Im Neuenheimer Feld 460, 69120, Heidelberg, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Seitz
authors.affiliation: STRATIFYER Molecular Pathology GmbH, Werthmannstr. 1c, 50935, Köln, Germany.
authors.forename: Sotirios
authors.initials: S
authors.lastname: Lakis
authors.affiliation: STRATIFYER Molecular Pathology GmbH, Werthmannstr. 1c, 50935, Köln, Germany.
authors.forename: Elke
authors.initials: E
authors.lastname: Veltrup
authors.affiliation: German Cancer Research Center, Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Altevogt
authors.affiliation: Department of Pathology, St. Elisabeth-Krankenhaus, Werthmannstr. 1c, 50935, Köln, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Eidt
authors.affiliation: STRATIFYER Molecular Pathology GmbH, Werthmannstr. 1c, 50935, Köln, Germany.|Department of Pathology, St. Elisabeth-Krankenhaus, Werthmannstr. 1c, 50935, Köln, Germany.
authors.forename: Ralph M
authors.initials: RM
authors.lastname: Wirtz
authors.affiliation: National Center for Tumor Diseases, University-Hospital Heidelberg, Im Neuenheimer Feld 460, 69120, Heidelberg, Germany.
authors.forename: Frederik
authors.initials: F
authors.lastname: Marmé
mesh_terms: D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use; D001943:Breast Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D018270:Carcinoma, Ductal, Breast / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D018275:Carcinoma, Lobular / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D017322:Clinical Trials, Phase II as Topic; D005260:Female; D006801:Humans; D007124:Immunoenzyme Techniques; D019394:Ki-67 Antigen / Q000235:genetics / Q000378:metabolism*; D020360:Neoadjuvant Therapy; D060787:Neoplasm Grading; D009367:Neoplasm Staging; D011379:Prognosis; D012333:RNA, Messenger / Q000235:genetics; D012372:ROC Curve; D016032:Randomized Controlled Trials as Topic; D060888:Real-Time Polymerase Chain Reaction; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism*; D011980:Receptors, Progesterone / Q000235:genetics / Q000378:metabolism*
chemical_list: D019394:Ki-67 Antigen; D012333:RNA, Messenger; D018719:Erb-b2 Receptor Tyrosine Kinases; D011960:Receptors, Estrogen; D011980:Receptors, Progesterone
keywords: Breast cancer; Image analysis; Immunohistochemistry (IHC); Ki67; MammaTyper®; Pathologic complete response; Prediction; RT-qPCR; mRNA; neoadjuvant