Virchows Archiv

, Volume 463, Issue 6, pp 775–786 | Cite as

Reliable PCR quantitation of estrogen, progesterone and ERBB2 receptor mRNA from formalin-fixed, paraffin-embedded tissue is independent of prior macro-dissection

  • Trine TrammEmail author
  • Guido Hennig
  • Marianne Kyndi
  • Jan Alsner
  • Flemming Brandt Sørensen
  • Simen Myhre
  • Therese Sørlie
  • Jens Overgaard
Original Article


Gene expression analysis on messenger RNA (mRNA) purified from formalin-fixed, paraffin-embedded tissue is increasingly used for research purposes. Tissue heterogeneity may question specificity and interpretation of results from mRNA isolated from a whole slide section, and thresholds for minimal tumor content in the paraffin block or macrodissection are used to avoid contamination from non-neoplastic tissue. The aim was to test if mRNA from tissue surrounding breast cancer affected quantification of estrogen receptor α (ESR1), progesterone receptor (PGR) and human epidermal growth factor receptor 2 (ERBB2), by comparing gene expression from whole slide and tumor-enriched sections, and correlating gene expression from whole slide sections with corresponding immunohistochemistry. Gene expression, based on mRNA extracted from a training set (36 paraffin blocks) and two validation sets (133 + 1,083 blocks), were determined by quantitative reverse transcription polymerase chain reaction for all samples, as well as by microarray for 133 validation samples. In the training set, agreement between high vs. low mRNA expression from whole slide and tumor-enriched sections was absolute for ESR1 and ERBB2, and 83 % for PGR. Overall agreements, when comparing mRNA expression to immunohistochemistry, were 100 % (ERBB2), 89 % (ESR1) and 83 % (PGR), which was confirmed in the validation sets. Percentage of tumor in the sections did not influence the results. In conclusion, reliable quantification of ESR1, PGR and ERBB2 mRNA expression can be obtained from a whole slide section, and correlates well with immunohistochemistry. Prior removal of surrounding tissue was found to be unnecessary even with minimal tumor content in the section.


Breast cancer Formalin fixed Immunohistochemistry Macrodissection Paraffin embedded Quantitative RT-PCR 



This study was supported by the Danish Cancer Society, and the Lundbeck Foundation Center for Interventional Research in Radiation Oncology (CIRRO); Fritz, Georg and Marie Cecilie Gluds Foundation; Helga and Peter Korning Foundation; Max and Inger Wørzners Memorial Foundation; Fonden til Lægevidenskabens Fremme (the A. P. Møller Foundation); Danish Agency for Science, Technology and Innovation; The Danish Research Council; and Aarhus University. We would like to thank Torsten Acht for the excellent technical work

Conflict of interest

Dr. Guido Hennig is an employee of Siemens Healthcare Diagnostics Holding GmbH, Eschborn, Germany. The authors declare that they have no other conflict of interest.

Supplementary material

428_2013_1486_MOESM1_ESM.pdf (166 kb)
Online resource 1 Table that presents the total data for the training set. (PDF 166 kb)
428_2013_1486_MOESM2_ESM.pdf (274 kb)
Online resource 2 Table that lists the histopathological characteristic of the training set. (PDF 274 kb)
428_2013_1486_MOESM3_ESM.pdf (257 kb)
Online resource 3 Bar plot showing the fraction of sections/cores with successful quantification of the four genes (RPL37A, ESR1, ERBB2, PGR). (PDF 256 kb)
428_2013_1486_MOESM4_ESM.pdf (217 kb)
Online resource 4 Histogram showing the distribution of tumor area fractions in the included 1,252 samples. (PDF 217 kb)
428_2013_1486_MOESM5_ESM.pdf (338 kb)
Online resource 5 Scatter plots showing the agreement between normalized gene expression from cores and sections of the same tumor block. (PDF 338 kb)
428_2013_1486_MOESM6_ESM.pdf (385 kb)
Online resource 6 Table that shows the number of discrepant samples between predictions of hormone and ERBB2 status from immunohistochemistry (IHC) and RT-qPCR. (PDF 384 kb)
428_2013_1486_MOESM7_ESM.pdf (358 kb)
Online resource 7 Scatter plots showing the correlation between numerical biochemical measurements and gene expression level from a whole slide section for ESR1 and PGR. (PDF 357 kb)
428_2013_1486_MOESM8_ESM.pdf (488 kb)
Online resource 8 Table that lists the agreements between biochemistry and IHC/RT-qPCR measurements. (PDF 487 kb)
428_2013_1486_MOESM9_ESM.pdf (493 kb)
Online resource 9 Table that shows the number of discrepant samples between predictions of hormone and ERBB2 status from microarray and IHC/RT-qPCR. (PDF 492 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Trine Tramm
    • 1
    Email author
  • Guido Hennig
    • 2
  • Marianne Kyndi
    • 1
    • 3
  • Jan Alsner
    • 1
  • Flemming Brandt Sørensen
    • 4
  • Simen Myhre
    • 5
    • 6
  • Therese Sørlie
    • 6
  • Jens Overgaard
    • 1
  1. 1.Department of Experimental Clinical OncologyAarhus University HospitalAarhusDenmark
  2. 2.Siemens Healthcare Diagnostics Holding GmbHEschbornGermany
  3. 3.Department of RadiologyAarhus University HospitalAarhusDenmark
  4. 4.Department of Clinical PathologyVejle HospitalVejleDenmark
  5. 5.Atlantis Medical University CollegeOsloNorway
  6. 6.Department of Genetics, Institute for Cancer ResearchOslo University HospitalOsloNorway

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