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Breast Cancer Research and Treatment

, Volume 138, Issue 1, pp 59–67 | Cite as

The detection of ESR1/PGR/ERBB2 mRNA levels by RT-QPCR: a better approach for subtyping breast cancer and predicting prognosis

  • Xin Du
  • Xiao-Qing Li
  • Lin Li
  • Yuan-Yuan Xu
  • Yu-Mei Feng
Preclinical study

Abstract

The molecular classification of breast cancer mainly focuses on ER, PR, and HER2 status detected by immunohistochemistry (IHC) analysis. To explore the clinical value of breast cancer classification based on gene-based diagnosis of the triple markers, we measured ESR1, PGR, and ERBB2 mRNA levels in 294 breast cancer patients by reverse transcription quantitative polymerase chain reaction (RT-QPCR), and examined their correlation with ER, PR, and HER2 status detected by IHC. We observed a significant positive correlation between the mRNA levels of the triple markers and their protein status (ESR1 vs. ER, Spearman’s ρ = 0.527, P = 2.3 × 10−22; PGR vs. PR, Spearman’s ρ = 0.631, P = 5.1 × 10−34; ERBB2 vs. HER2, Spearman’s ρ = 0.439, P = 3.0 × 10−15). Furthermore, the subtypes determined by mRNA levels of the triple markers were significantly correlated to the subtypes determined based on their protein status (Spearman’s ρ = 0.342, P = 2.0 × 10−8). Kaplan–Meier analysis showed that the subtypes determined by mRNA levels of the triple-marker could predict the disease-free survival (DFS) in breast cancer patients. Multivariate analysis showed that the predictive value of DFS could be confirmed for the subtypes determined by mRNA levels of the triple markers (HR = 2.285, P = 0.008) but not for those determined by their protein status. Taken together, our results suggest that the detection of ESR1/PGR/ERBB2 mRNA levels by RT-QPCR is a better approach for subtyping breast cancer and predicting the prognosis.

Keywords

RT-QPCR Molecular subtype Breast cancer Prognosis 

Notes

Acknowledgments

This study was supported by the National Natural Science Foundation of China (No. 30872518 and No. 81272357), the Applied Basic Research Projects of Tianjin (No. 06YFJMJC12900 and No. 09JCZDJC19800).

Conflict of interests

The authors declare that they have no conflict of interest.

References

  1. 1.
    DeSantis C, Siegel R, Bandi P, Jemal A (2011) Breast cancer statistics. CA Cancer J Clin 61(6):409–418PubMedCrossRefGoogle Scholar
  2. 2.
    Parker JS, Mullins M, Cheang MC, Leung S, Voduc D, Vickery T, Davies S, Fauron C, He X, Hu Z, Quackenbush JF, Stijleman IJ, Palazzo J, Marron JS, Nobel AB, Mardis E, Nielsen TO, Ellis MJ, Perou CM, Bernard PS (2009) Supervised risk predictor of breast cancer based on intrinsic subtypes. J Clin Oncol 27(8):1160–1167PubMedCrossRefGoogle Scholar
  3. 3.
    Perou CM, Sorlie T, Eisen MB, van de Rijn M, Jeffrey SS, Rees CA, Pollack JR, Ross DT, Johnsen H, Akslen LA, Fluge O, Pergamenschikov A, Williams C, Zhu SX, Lonning PE, Borresen-Dale AL, Brown PO, Botstein D (2000) Molecular portraits of human breast tumours. Nature 406(6797):747–752PubMedCrossRefGoogle Scholar
  4. 4.
    Nguyen PL, Taghian AG, Katz MS, Niemierko A, Abi Raad RF, Boon WL, Bellon JR, Wong JS, Smith BL, Harris JR (2008) Breast cancer subtype approximated by estrogen receptor, progesterone receptor, and HER-2 is associated with local and distant recurrence after breast-conserving therapy. J Clin Oncol 26(14):2373–2378PubMedCrossRefGoogle Scholar
  5. 5.
    Nielsen TO, Hsu FD, Jensen K, Cheang M, Karaca G, Hu Z, Hernandez-Boussard T, Livasy C, Cowan D, Dressler L, Akslen LA, Ragaz J, Gown AM, Gilks CB, van de Rijn M, Perou CM (2004) Immunohistochemical and clinical characterization of the basal-like subtype of invasive breast carcinoma. Clin Cancer Res 10(16):5367–5374PubMedCrossRefGoogle Scholar
  6. 6.
    Irvin WJ Jr, Carey LA (2008) What is triple-negative breast cancer? Eur J Cancer 44(18):2799–2805PubMedCrossRefGoogle Scholar
  7. 7.
    van’t Veer LJ, Dai H, van de Vijver MJ, He YD, Hart AA, Mao M, Peterse HL, van der Kooy K, Marton MJ, Witteveen AT, Schreiber GJ, Kerkhoven RM, Roberts C, Linsley PS, Bernards R, Friend SH (2002) Gene expression profiling predicts clinical outcome of breast cancer. Nature 415(6871):530–536CrossRefGoogle Scholar
  8. 8.
    Sotiriou C, Neo SY, McShane LM, Korn EL, Long PM, Jazaeri A, Martiat P, Fox SB, Harris AL, Liu ET (2003) Breast cancer classification and prognosis based on gene expression profiles from a population-based study. Proc Natl Acad Sci USA 100(18):10393–10398PubMedCrossRefGoogle Scholar
  9. 9.
    Sorlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H, Hastie T, Eisen MB, van de Rijn M, Jeffrey SS, Thorsen T, Quist H, Matese JC, Brown PO, Botstein D, Eystein Lonning P, Borresen-Dale AL (2001) Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci USA 98(19):10869–10874PubMedCrossRefGoogle Scholar
  10. 10.
    Liedtke C, Mazouni C, Hess KR, Andre F, Tordai A, Mejia JA, Symmans WF, Gonzalez-Angulo AM, Hennessy B, Green M, Cristofanilli M, Hortobagyi GN, Pusztai L (2008) Response to neoadjuvant therapy and long-term survival in patients with triple-negative breast cancer. J Clin Oncol 26(8):1275–1281PubMedCrossRefGoogle Scholar
  11. 11.
    Rouzier R, Perou CM, Symmans WF, Ibrahim N, Cristofanilli M, Anderson K, Hess KR, Stec J, Ayers M, Wagner P, Morandi P, Fan C, Rabiul I, Ross JS, Hortobagyi GN, Pusztai L (2005) Breast cancer molecular subtypes respond differently to preoperative chemotherapy. Clin Cancer Res 11(16):5678–5685PubMedCrossRefGoogle Scholar
  12. 12.
    Carey LA, Perou CM, Livasy CA, Dressler LG, Cowan D, Conway K, Karaca G, Troester MA, Tse CK, Edmiston S, Deming SL, Geradts J, Cheang MC, Nielsen TO, Moorman PG, Earp HS, Millikan RC (2006) Race, breast cancer subtypes, and survival in the Carolina Breast Cancer Study. JAMA 295(21):2492–2502PubMedCrossRefGoogle Scholar
  13. 13.
    Regan MM, Viale G, Mastropasqua MG, Maiorano E, Golouh R, Carbone A, Brown B, Suurkula M, Langman G, Mazzucchelli L, Braye S, Grigolato P, Gelber RD, Castiglione-Gertsch M, Price KN, Coates AS, Goldhirsch A, Gusterson B (2006) Re-evaluating adjuvant breast cancer trials: assessing hormone receptor status by immunohistochemical versus extraction assays. J Natl Cancer Inst 98(21):1571–1581PubMedCrossRefGoogle Scholar
  14. 14.
    Rhodes A, Jasani B, Barnes DM, Bobrow LG, Miller KD (2000) Reliability of immunohistochemical demonstration of oestrogen receptors in routine practice: interlaboratory variance in the sensitivity of detection and evaluation of scoring systems. J Clin Pathol 53(2):125–130PubMedCrossRefGoogle Scholar
  15. 15.
    Goldstein NS, Ferkowicz M, Odish E, Mani A, Hastah F (2003) Minimum formalin fixation time for consistent estrogen receptor immunohistochemical staining of invasive breast carcinoma. Am J Clin Pathol 120(1):86–92PubMedCrossRefGoogle Scholar
  16. 16.
    Press MF, Slamon DJ, Flom KJ, Park J, Zhou JY, Bernstein L (2002) Evaluation of HER-2/neu gene amplification and overexpression: comparison of frequently used assay methods in a molecularly characterized cohort of breast cancer specimens. J Clin Oncol 20(14):3095–3105PubMedGoogle Scholar
  17. 17.
    Roche PC, Suman VJ, Jenkins RB, Davidson NE, Martino S, Kaufman PA, Addo FK, Murphy B, Ingle JN, Perez EA (2002) Concordance between local and central laboratory HER2 testing in the breast intergroup trial N9831. J Natl Cancer Inst 94(11):855–857PubMedCrossRefGoogle Scholar
  18. 18.
    Wolff AC, Hammond ME, Schwartz JN, Hagerty KL, Allred DC, Cote RJ, Dowsett M, Fitzgibbons PL, Hanna WM, Langer A, McShane LM, Paik S, Pegram MD, Perez EA, Press MF, Rhodes A, Sturgeon C, Taube SE, Tubbs R, Vance GH, van de Vijver M, Wheeler TM, Hayes DF (2007) American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. J Clin Oncol 25(1):118–145PubMedCrossRefGoogle Scholar
  19. 19.
    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(1):35–42PubMedCrossRefGoogle Scholar
  20. 20.
    Petit T, Wilt M, Velten M, Millon R, Rodier JF, Borel C, Mors R, Haegele P, Eber M, Ghnassia JP (2004) Comparative value of tumour grade, hormonal receptors, Ki-67, HER-2 and topoisomerase II alpha status as predictive markers in breast cancer patients treated with neoadjuvant anthracycline-based chemotherapy. Eur J Cancer 40(2):205–211PubMedCrossRefGoogle Scholar
  21. 21.
    Tse C, Brault D, Gligorov J, Antoine M, Neumann R, Lotz JP, Capeau J (2005) Evaluation of the quantitative analytical methods real-time PCR for HER-2 gene quantification and ELISA of serum HER-2 protein and comparison with fluorescence in situ hybridization and immunohistochemistry for determining HER-2 status in breast cancer patients. Clin Chem 51(7):1093–1101PubMedCrossRefGoogle Scholar
  22. 22.
    van de Vijver MJ, He YD, van’t Veer LJ, Dai H, Hart AA, Voskuil DW, Schreiber GJ, Peterse JL, Roberts C, Marton MJ, Parrish M, Atsma D, Witteveen A, Glas A, Delahaye L, van der Velde T, Bartelink H, Rodenhuis S, Rutgers ET, Friend SH, Bernards R (2002) A gene-expression signature as a predictor of survival in breast cancer. N Engl J Med 347(25):1999–2009PubMedCrossRefGoogle Scholar
  23. 23.
    Barberis M, Pellegrini C, Cannone M, Arizzi C, Coggi G, Bosari S (2008) Quantitative PCR and HER2 testing in breast cancer: a technical and cost-effectiveness analysis. Am J Clin Pathol 129(4):563–570PubMedCrossRefGoogle Scholar
  24. 24.
    McShane LM, Altman DG, Sauerbrei W, Taube SE, Gion M, Clark GM (2006) REporting recommendations for tumor MARKer prognostic studies (REMARK). Breast Cancer Res Treat 100(2):229–235PubMedCrossRefGoogle Scholar
  25. 25.
    Li X, Cao X, Zhang W, Feng Y (2007) Expression level of insulin-like growth factor binding protein 5 mRNA is a prognostic factor for breast cancer. Cancer Sci 98(10):1592–1596PubMedCrossRefGoogle Scholar
  26. 26.
    Liu F, Lang R, Zhao J, Zhang X, Pringle GA, Fan Y, Yin D, Gu F, Yao Z, Fu L (2011) CD8(+) cytotoxic T cell and FOXP3(+) regulatory T cell infiltration in relation to breast cancer survival and molecular subtypes. Breast Cancer Res Treat 130(2):645–655PubMedCrossRefGoogle Scholar
  27. 27.
    Scott A, Ambannavar R, Jeong J, Liu ML, Cronin MT (2011) RT-PCR-based gene expression profiling for cancer biomarker discovery from fixed, paraffin-embedded tissues. Methods Mol Biol 724:239–257PubMedCrossRefGoogle Scholar
  28. 28.
    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):1–10PubMedCrossRefGoogle Scholar
  29. 29.
    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(2):117–130PubMedCrossRefGoogle Scholar
  30. 30.
    Noske A, Loibl S, Darb-Esfahani S, Roller M, Kronenwett R, Muller BM, Steffen J, von Toerne C, Wirtz R, Baumann I, Hoffmann G, Heinrich G, Grasshoff ST, Ulmer HU, Denkert C, von Minckwitz G (2011) Comparison of different approaches for assessment of HER2 expression on protein and mRNA level: prediction of chemotherapy response in the neoadjuvant GeparTrio trial (NCT00544765). Breast Cancer Res Treat 126(1):109–117PubMedCrossRefGoogle Scholar
  31. 31.
    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(28):4611–4619PubMedCrossRefGoogle Scholar
  32. 32.
    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(15):2473–2481PubMedCrossRefGoogle Scholar
  33. 33.
    Gobbi H, Dupont WD, Parl FF, Schuyler PA, Plummer WD, Olson SJ, Page DL (2005) Breast cancer risk associated with estrogen receptor expression in epithelial hyperplasia lacking atypia and adjacent lobular units. Int J Cancer 113(5):857–859PubMedCrossRefGoogle Scholar
  34. 34.
    Fitzgibbons PL, Page DL, Weaver D, Thor AD, Allred DC, Clark GM, Ruby SG, O’Malley F, Simpson JF, Connolly JL, Hayes DF, Edge SB, Lichter A, Schnitt SJ (2000) Prognostic factors in breast cancer. College of American Pathologists Consensus Statement 1999. Arch Pathol Lab Med 124(7):966–978PubMedGoogle Scholar
  35. 35.
    Diaz LK, Sahin A, Sneige N (2004) Interobserver agreement for estrogen receptor immunohistochemical analysis in breast cancer: a comparison of manual and computer-assisted scoring methods. Ann Diagn pathol 8(1):23–27PubMedCrossRefGoogle Scholar
  36. 36.
    Fisher ER, Anderson S, Dean S, Dabbs D, Fisher B, Siderits R, Pritchard J, Pereira T, Geyer C, Wolmark N (2005) Solving the dilemma of the immunohistochemical and other methods used for scoring estrogen receptor and progesterone receptor in patients with invasive breast carcinoma. Cancer 103(1):164–173PubMedCrossRefGoogle Scholar
  37. 37.
    Kraus JA, Dabbs DJ, Beriwal S, Bhargava R (2012) Semi-quantitative immunohistochemical assay versus oncotype DX((R)) qRT-PCR assay for estrogen and progesterone receptors: an independent quality assurance study. Mod Pathol 25(6):869–876PubMedCrossRefGoogle Scholar
  38. 38.
    Hahnel R, Woodings T, Vivian AB (1979) Prognostic value of estrogen receptors in primary breast cancer. Cancer 44(2):671–675PubMedCrossRefGoogle Scholar
  39. 39.
    Gown AM (2008) Current issues in ER and HER2 testing by IHC in breast cancer. Mod Pathol 21(Suppl 2):S8–S15PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Xin Du
    • 1
  • Xiao-Qing Li
    • 1
    • 2
  • Lin Li
    • 1
  • Yuan-Yuan Xu
    • 1
  • Yu-Mei Feng
    • 1
    • 2
  1. 1.Department of Biochemistry and Molecular BiologyTianjin Medical University Cancer Institute and HospitalTianjinChina
  2. 2.Key Laboratory of Breast Cancer Prevention and Treatment of the Ministry of EducationTianjin Medical University Cancer Institute and HospitalTianjinChina

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