Quantitative in situ measurement of estrogen receptor mRNA predicts response to tamoxifen

PLoS One. 2012;7(5):e36559. doi: 10.1371/journal.pone.0036559. Epub 2012 May 11.


Purpose: Quantification of mRNA has historically been done by reverse transcription polymerase chain reaction (RT-PCR). Recently, a robust method of detection of mRNA utilizing in situ hybridization has been described that is linear and shows high specificity with low background. Here we describe the use of the AQUA method of quantitative immunofluorescence (QIF) for measuring mRNA in situ using ESR1 (the estrogen receptor alpha gene) in breast cancer to determine its predictive value compared to Estrogen Receptor α (ER) protein.

Methods: Messenger RNA for ER (ESR1) and Ubiquitin C (UbC) were visualized using RNAscope probes and levels were quantified by quantitative in situ hybridization (qISH) on two Yale breast cancer cohorts on tissue microarrays. ESR1 levels were compared to ER protein levels measured by QIF using the SP1 antibody.

Results: ESR1 mRNA is reproducibly and specifically measurable by qISH on tissue collected from 1993 or later. ESR1 levels were correlated to ER protein levels in a non-linear manner on two Yale cohorts. High levels of ESR1 were found to be predictive of response to tamoxifin.

Conclusion: Quantification of mRNA using qISH may allow assessment of large cohorts with minimal formalin fixed, paraffin embedded tissue. Exploratory data using this method suggests that measurement of ESR1 mRNA levels may be predictive of response to endocrine therapy in a manner that is different from the predictive value of ER.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Validation Study

MeSH terms

  • Adult
  • Aged
  • Antineoplastic Agents, Hormonal / therapeutic use
  • Breast Neoplasms / drug therapy*
  • Breast Neoplasms / genetics*
  • Breast Neoplasms / metabolism
  • Cohort Studies
  • Estrogen Receptor Modulators / therapeutic use
  • Estrogen Receptor alpha / genetics*
  • Estrogen Receptor alpha / metabolism
  • Female
  • Humans
  • In Situ Hybridization / methods
  • Middle Aged
  • Predictive Value of Tests
  • RNA, Messenger / genetics*
  • RNA, Messenger / metabolism*
  • RNA, Neoplasm / genetics*
  • RNA, Neoplasm / metabolism*
  • Tamoxifen / therapeutic use*


  • Antineoplastic Agents, Hormonal
  • Estrogen Receptor Modulators
  • Estrogen Receptor alpha
  • RNA, Messenger
  • RNA, Neoplasm
  • estrogen receptor alpha, human
  • Tamoxifen