Advertisement

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

Abstract

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.

Keywords

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

Notes

Acknowledgments

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)

References

  1. 1.
    Carlson RW, Allred DC, Anderson BO, Burstein HJ, Carter WB, Edge SB, Erban JK, Farrar WB, Goldstein LJ, Gradishar WJ, Hayes DF, Hudis CA, Jahanzeb M, Kiel K, Ljung BM, Marcom PK, Mayer IA, McCormick B, Nabell LM, Pierce LJ, Reed EC, Smith ML, Somlo G, Theriault RL, Topham NS, Ward JH, Winer EP, Wolff AC, NCCN Breast Cancer Clinical Practice Guidelines Panel (2009) Breast cancer. Clinical practice guidelines in oncology. J Natl Compr Canc Netw 7:122–192PubMedGoogle Scholar
  2. 2.
    Goldhirsch A, Wood WC, Coates AS, Gelber RD, Thurlimann B, Senn HJ, Panel members (2011) Strategies for subtypes—dealing with the diversity of breast cancer: highlights of the St. Gallen international expert consensus on the primary therapy of early breast cancer 2011. Ann Oncol 22:1736–1747PubMedCrossRefGoogle Scholar
  3. 3.
    Bohmann K, Hennig G, Rogel U, Poremba C, Mueller BM, Fritz P, Stoerkel S, Schaefer KL (2009) RNA extraction from archival formalin-fixed paraffin-embedded tissue: a comparison of manual, semiautomated, and fully automated purification methods. Clin Chem 55:1719–1727PubMedCrossRefGoogle Scholar
  4. 4.
    Hennig G, Gehrmann M, Stropp U, Brauch H, Fritz P, Eichelbaum M, Schwab M, Schroth W (2010) Automated extraction of DNA and RNA from a single formalin-fixed paraffin-embedded tissue section for analysis of both single-nucleotide polymorphisms and mRNA expression. Clin Chem 56:1845–1853PubMedCrossRefGoogle Scholar
  5. 5.
    Muller BM, Kronenwett R, Hennig G, Euting H, Weber K, Bohmann K, Weichert W, Altmann G, Roth C, Winzer KJ, Kristiansen G, Petry C, Dietel M, Denkert C (2011) Quantitative determination of estrogen receptor, progesterone receptor, and HER2 mRNA in formalin-fixed paraffin-embedded tissue—a new option for predictive biomarker assessment in breast cancer. Diagn Mol Pathol 20:1–10PubMedCrossRefGoogle Scholar
  6. 6.
    Badve SS, Baehner FL, Gray RP, Childs BH, Maddala T, Liu ML, Rowley SC, Shak S, Perez EA, Shulman LJ, Martino S, Davidson NE, Sledge GW, Goldstein LJ, Sparano JA (2008) Estrogen- and progesterone-receptor status in ECOG 2197: comparison of immunohistochemistry by local and central laboratories and quantitative reverse transcription polymerase chain reaction by central laboratory. J Clin Oncol 26:2473–2481PubMedCrossRefGoogle Scholar
  7. 7.
    Antonov J, Popovici V, Delorenzi M, Wirapati P, Baltzer A, Oberli A, Thurlimann B, Giobbie-Hurder A, Viale G, Altermatt HJ, Aebi S, Jaggi R (2010) Molecular risk assessment of BIG 1–98 participants by expression profiling using RNA from archival tissue. BMC Cancer 10:37PubMedCrossRefGoogle Scholar
  8. 8.
    Iverson AA, Gillett C, Cane P, Santini CD, Vess TM, Kam-Morgan L, Wang A, Eisenberg M, Rowland CM, Hessling JJ, Broder SE, Sninsky JJ, Tutt A, Anderson S, Chang SY (2009) A single-tube quantitative assay for mRNA levels of hormonal and growth factor receptors in breast cancer specimens. J Mol Diagn 11:117–130PubMedCrossRefGoogle Scholar
  9. 9.
    Paik S, Shak S, Tang G, Kim C, Baker J, Cronin M, Baehner FL, Walker MG, Watson D, Park T, Hiller W, Fisher ER, Wickerham DL, Bryant J, Wolmark N (2004) A multigene assay to predict recurrence of tamoxifen-treated, node-negative breast cancer. N Engl J Med 351:2817–2826PubMedCrossRefGoogle Scholar
  10. 10.
    Kyndi M, Sorensen FB, Knudsen H, Overgaard M, Nielsen HM, Andersen J, Overgaard J (2008) Tissue microarrays compared with whole sections and biochemical analyses. A subgroup analysis of DBCG 82 b&c. Acta Oncol 47:591–599PubMedCrossRefGoogle Scholar
  11. 11.
    Kyndi M, Sorensen FB, Knudsen H, Overgaard M, Nielsen HM, Overgaard J, Danish Breast Cancer Cooperative Group (2008) Estrogen receptor, progesterone receptor, HER-2, and response to postmastectomy radiotherapy in high-risk breast cancer: the Danish Breast Cancer Cooperative Group. J Clin Oncol 26:1419–1426PubMedCrossRefGoogle Scholar
  12. 12.
    Jensen AR, Storm HH, Moller S, Overgaard J (2003) Validity and representativity in the danish breast cancer cooperative group—a study on protocol allocation and data validity from one county to a multi-centre database. Acta Oncol 42:179–185PubMedCrossRefGoogle Scholar
  13. 13.
    Myhre S, Mohammed H, Tramm T, Alsner J, Finak G, Park M, Overgaard J, Borresen-Dale AL, Frigessi A, Sorlie T (2010) In silico ascription of gene expression differences to tumor and stromal cells in a model to study impact on breast cancer outcome. PLoS One 5:e14002PubMedCrossRefGoogle Scholar
  14. 14.
    Andersen J, Bentzen SM, Poulsen HS (1988) Relationship between radioligand binding assay, immunoenzyme assay and immunohistochemical assay for estrogen receptors in human breast cancer and association with tumor differentiation. Eur J Cancer Clin Oncol 24:377–384PubMedCrossRefGoogle Scholar
  15. 15.
    Gundersen HJ, Bendtsen TF, Korbo L, Marcussen N, Moller A, Nielsen K, Nyengaard JR, Pakkenberg B, Sorensen FB, Vesterby A (1988) Some new, simple and efficient stereological methods and their use in pathological research and diagnosis. APMIS 96:379–394PubMedCrossRefGoogle Scholar
  16. 16.
    Toustrup K, Sorensen BS, Lassen P, Wiuf C, Alsner J, Overgaard J, On behalf of the Danish Head and Neck Cancer Group (DAHANCA) (2012) Gene expression classifier predicts for hypoxic modification of radiotherapy with nimorazole in squamous cell carcinomas of the head and neck. Radiother Oncol 102:122–129PubMedCrossRefGoogle Scholar
  17. 17.
    Filipits M, Rudas M, Jakesz R, Dubsky P, Fitzal F, Singer CF, Dietze O, Greil R, Jelen A, Sevelda P, Freibauer C, Muller V, Janicke F, Schmidt M, Kolbl H, Rody A, Kaufmann M, Schroth W, Brauch H, Schwab M, Fritz P, Weber KE, Feder IS, Hennig G, Kronenwett R, Gehrmann M, Gnant M, Investigators EP (2011) A new molecular predictor of distant recurrence in ER-positive, HER2-negative breast cancer adds independent information to conventional clinical risk factors. Clin Cancer Res 17:6012–6020PubMedCrossRefGoogle Scholar
  18. 18.
    Tramm T, Sorensen BS, Overgaard J, Alsner J (2013) Optimal reference genes for normalization of qRT-PCR data from archival formalin-fixed, paraffin-embedded breast tumors controlling for tumor cell content and decay of mRNA. Diagn Mol Pathol 22:181–187PubMedCrossRefGoogle Scholar
  19. 19.
    Myhre S, Lingjaerde OC, Hennessy BT, Aure MR, Carey MS, Alsner J, Tramm T, Overgaard J, Mills GB, Borresen-Dale AL, Sorlie T (2013) Influence of DNA copy number and mRNA levels on the expression of breast cancer related proteins. Mol Oncol 7(3):704–18Google Scholar
  20. 20.
    Cronin M, Pho M, Dutta D, Stephans JC, Shak S, Kiefer MC, Esteban JM, Baker JB (2004) Measurement of gene expression in archival paraffin-embedded tissues: development and performance of a 92-gene reverse transcriptase-polymerase chain reaction assay. Am J Pathol 164:35–42PubMedCrossRefGoogle Scholar
  21. 21.
    Ma XJ, Hilsenbeck SG, Wang W, Ding L, Sgroi DC, Bender RA, Osborne CK, Allred DC, Erlander MG (2006) The HOXB13:IL17BR expression index is a prognostic factor in early-stage breast cancer. J Clin Oncol 24:4611–4619PubMedCrossRefGoogle Scholar
  22. 22.
    Mina L, Soule SE, Badve S, Baehner FL, Baker J, Cronin M, Watson D, Liu ML, Sledge GW Jr, Shak S, Miller KD (2007) Predicting response to primary chemotherapy: gene expression profiling of paraffin-embedded core biopsy tissue. Breast Cancer Res Treat 103:197–208PubMedCrossRefGoogle Scholar
  23. 23.
    Paik S, Kim CY, Song YK, Kim WS (2005) Technology insight: application of molecular techniques to formalin-fixed paraffin-embedded tissues from breast cancer. Nat Clin Pract Oncol 2:246–254PubMedCrossRefGoogle Scholar
  24. 24.
    Gong Y, Yan K, Lin F, Anderson K, Sotiriou C, Andre F, Holmes FA, Valero V, Booser D, Pippen JE Jr, Vukelja S, Gomez H, Mejia J, Barajas LJ, Hess KR, Sneige N, Hortobagyi GN, Pusztai L, Symmans WF (2007) Determination of oestrogen-receptor status and ERBB2 status of breast carcinoma: a gene-expression profiling study. Lancet Oncol 8:203–211PubMedCrossRefGoogle Scholar
  25. 25.
    Roepman P, Horlings HM, Krijgsman O, Kok M, Bueno-de-Mesquita JM, Bender R, Linn SC, Glas AM, van de Vijver MJ (2009) Microarray-based determination of estrogen receptor, progesterone receptor, and HER2 receptor status in breast cancer. Clin Cancer Res 15:7003–7011PubMedCrossRefGoogle Scholar
  26. 26.
    Pusztai L, Viale G, Kelly CM, Hudis CA (2010) Estrogen and HER-2 receptor discordance between primary breast cancer and metastasis. Oncologist 15:1164–1168PubMedCrossRefGoogle Scholar
  27. 27.
    Talman ML, Rasmussen BB, Andersen J, Christensen IJ (2008) Estrogen receptor analyses in the danish breast cancer cooperative group. History, methods, prognosis and clinical implications. Acta Oncol 47:789–794PubMedCrossRefGoogle Scholar
  28. 28.
    Le Romancer M, Poulard C, Cohen P, Sentis S, Renoir JM, Corbo L (2011) Cracking the estrogen receptor's posttranslational code in breast tumors. Endocr Rev 32:597–622PubMedCrossRefGoogle Scholar
  29. 29.
    Chebil G, Bendahl PO, Ferno M, South Sweden Breast Cancer Group, North Sweden Breast Cancer Group (2003) Estrogen and progesterone receptor assay in paraffin-embedded breast cancer—reproducibility of assessment. Acta Oncol 42:43–47PubMedCrossRefGoogle Scholar
  30. 30.
    Cserni G, Francz M, Kalman E, Kelemen G, Komjathy DC, Kovacs I, Kulka J, Sarkadi L, Udvarhelyi N, Vass L, Voros A (2011) Estrogen receptor negative and progesterone receptor positive breast carcinomas-how frequent are they? Pathol Oncol Res 17:663–668PubMedCrossRefGoogle Scholar
  31. 31.
    Rakha EA, El-Sayed ME, Green AR, Paish EC, Powe DG, Gee J, Nicholson RI, Lee AH, Robertson JF, Ellis IO (2007) Biologic and clinical characteristics of breast cancer with single hormone receptor positive phenotype. J Clin Oncol 25:4772–4778PubMedCrossRefGoogle Scholar
  32. 32.
    Hefti MM, Hu R, Knoblauch NW, Collins LC, Haibe-Kains B, Tamimi RM, Beck AH (2013) Estrogen receptor negative/progesterone receptor positive breast cancer is not a reproducible subtype. Breast Cancer Res 15:R68PubMedCrossRefGoogle Scholar
  33. 33.
    Harvey JM, Clark GM, Osborne CK, Allred DC (1999) Estrogen receptor status by immunohistochemistry is superior to the ligand-binding assay for predicting response to adjuvant endocrine therapy in breast cancer. J Clin Oncol 17:1474–1481PubMedGoogle Scholar
  34. 34.
    Viale G, Regan MM, Maiorano E, Mastropasqua MG, Golouh R, Perin T, Brown RW, Kovacs A, Pillay K, Ohlschlegel C, Braye S, Grigolato P, Rusca T, Gelber RD, Castiglione-Gertsch M, Price KN, Goldhirsch A, Gusterson BA, Coates AS (2008) Chemoendocrine compared with endocrine adjuvant therapies for node-negative breast cancer: predictive value of centrally reviewed expression of estrogen and progesterone receptors—international breast cancer study group. J Clin Oncol 26:1404–1410PubMedCrossRefGoogle Scholar
  35. 35.
    Viale G, Regan MM, Maiorano E, Mastropasqua MG, Dell'Orto P, Rasmussen BB, Raffoul J, Neven P, Orosz Z, Braye S, Ohlschlegel C, Thurlimann B, Gelber RD, Castiglione-Gertsch M, Price KN, Goldhirsch A, Gusterson BA, Coates AS (2007) Prognostic and predictive value of centrally reviewed expression of estrogen and progesterone receptors in a randomized trial comparing letrozole and tamoxifen adjuvant therapy for postmenopausal early breast cancer: BIG 1–98. J Clin Oncol 25:3846–3852PubMedCrossRefGoogle Scholar

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

Personalised recommendations