Comparison of immunohistochemistry with PCR for assessment of ER, PR, and Ki-67 and prediction of pathological complete response in breast cancer

BMC Cancer. 2017 Feb 13;17(1):124. doi: 10.1186/s12885-017-3111-1.


Background: 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.

Methods: 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.

Results: 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).

Conclusion: 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.

Keywords: Breast cancer; Image analysis; Immunohistochemistry (IHC); Ki67; MammaTyper®; Pathologic complete response; Prediction; RT-qPCR; mRNA; neoadjuvant.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antineoplastic Combined Chemotherapy Protocols / therapeutic use
  • Breast Neoplasms / drug therapy
  • Breast Neoplasms / genetics
  • Breast Neoplasms / metabolism
  • Breast Neoplasms / pathology*
  • Carcinoma, Ductal, Breast / drug therapy
  • Carcinoma, Ductal, Breast / genetics
  • Carcinoma, Ductal, Breast / metabolism
  • Carcinoma, Ductal, Breast / pathology*
  • Carcinoma, Lobular / drug therapy
  • Carcinoma, Lobular / genetics
  • Carcinoma, Lobular / metabolism
  • Carcinoma, Lobular / pathology*
  • Clinical Trials, Phase II as Topic
  • Female
  • Humans
  • Immunoenzyme Techniques
  • Ki-67 Antigen / genetics
  • Ki-67 Antigen / metabolism*
  • Neoadjuvant Therapy
  • Neoplasm Grading
  • Neoplasm Staging
  • Prognosis
  • RNA, Messenger / genetics
  • ROC Curve
  • Randomized Controlled Trials as Topic
  • Real-Time Polymerase Chain Reaction
  • Receptor, ErbB-2 / genetics
  • Receptor, ErbB-2 / metabolism
  • Receptors, Estrogen / genetics
  • Receptors, Estrogen / metabolism*
  • Receptors, Progesterone / genetics
  • Receptors, Progesterone / metabolism*


  • Ki-67 Antigen
  • RNA, Messenger
  • Receptors, Estrogen
  • Receptors, Progesterone
  • Receptor, ErbB-2