Breast Cancer

, Volume 25, Issue 4, pp 416–430 | Cite as

Clinical utility of RT-PCR in assessing HER 2 gene expression versus traditional IHC and FISH in breast cancer patients

  • Moushumi Suryavanshi
  • Anurag Mehta
  • Jiten Jaipuria
  • Dushyant Kumar
  • Gayatri Vishwakarma
  • Manoj Kumar Panigrahi
  • Haristuti Verma
  • Mumtaz Saifi
  • Sanjeev Sharma
  • Simran Tandon
  • D. C. Doval
  • Bhudev C. Das
Original Article



IHC and FISH are used for categorizing HER 2 status in breast cancer at the protein and DNA level, respectively. HER 2 expression at the RNA level is quantitative, cheaper, easier to standardize and free from interobserver variation.


115 consecutive patients were tested by IHC, FISH and RT-PCR (test cohort). Assuming FISH result to be the response variable, ROC curves for RT-PCR ratio were analyzed to label HER 2 negative, equivocal and positive cases as RT-PCR score 1, 2 and 3, respectively. Inter-relationships between RT-PCR, IHC and FISH were defined. ‘Clinical benefit’ of a test was defined as proportion of patients labeled unequivocally as HER 2 positive or negative. Population for 1 year was simulated constraint to previous reports of HER 2 positivity and IHC category distribution by a meta-analysis of previous studies that evaluated concordance between IHC and FISH to determine HER 2 status (simulation cohort). Four diagnostic pathways in the simulation cohort were defined—(1) initial IHC, followed by FISH (conventional pathway); (2) initial RT-PCR, followed by FISH; (3) initial IHC, followed by RT-PCR and then by FISH; (4) initial RT-PCR, followed by IHC and then by FISH. The clinical benefit of IHC and RT-PCR in the four pathways was analyzed and sensitivity analysis for incremental cost-effectiveness ratio and cost–benefit comapring RT-PCR against IHC, both as first-line tests and among those with IHC score 2 as a reflex second-line test was performed by the Monte Carlo technique.


115 patients comprised the study population. While none with IHC score of 0 or 1 was FISH positive for HER 2, all cases with IHC score of 3 were FISH positive. 43 cases were assigned IHC score of 2. Thus, 72 patients benefited from the initial IHC testing [clinical benefit 62.6%], with the overall concordance between IHC and FISH being 100% for those with IHC score of 0, 1 and 3 (conclusive IHC categories). For RT-PCR with 100% concordance, 15.7% (115–97 = 18) patients would have benefited from RT-PCR testing if it was used as a first-line test. If RT-PCR would have been used as a second-line test among those with IHC score 2 (n = 43), then only 6 patients would have been assigned a conclusive RT-PCR category (category 1 or 3) translating to a clinical benefit of 14% (6/43) as a second-line test. As a second-line test it had 51% probability to prove more cost-effective than the conventional pathway, provided the cost of RT-PCR was 0.4 times the cost of IHC. Also in a three-step pathway, RT-PCR upfront would have 56% probability of higher cost–benefit provided the cost of RT-PCR was 0.1 times the cost of IHC.


RT-PCR results were found to be suboptimal to IHC in terms of discriminative ability and clinical benefit; thus, it is unlikely to replace IHC as a first-line test in the near future.


HER 2 RT-PCR Breast cancer Clinical benefit 


Author contribution

All authors equally contributed to manuscript designing, analysis and editing.


Institutional funded study.

Compliance with ethical standards

Conflict of interest

The authors declare that they do not have any conflict of interest.


  1. 1.
    Hurvitz SA, Hu Y, O’Brien N, Finn RS. Current approaches and future directions in the treatment of HER2-positive breast cancer. Cancer Treat Rev. 2013;39(3):219–29.CrossRefPubMedGoogle Scholar
  2. 2.
    Levy A, Borget I, Bahri M, Arnedos M, Rivin E, Vielh P, et al. Loco-regional control after neo-adjuvant chemotherapy and conservative treatment for locally advanced breast cancer patients. Breast J. 2014;20(4):381–7.CrossRefPubMedGoogle Scholar
  3. 3.
    Perez EA, Romond EH, Suman VJ, Jeong JH, Sledge G, Geyer CE Jr, et al. Trastuzumab plus adjuvant chemotherapy for human epidermal growth factor receptor 2-positive breast cancer: planned joint analysis of overall survival from NSABP B-31 and NCCTG N9831. J Clin Oncol. 2014;32(33):3744–52.CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Wolff AC, Hammond ME, Hicks DG, Dowsett M, McShane LM, Allison KH, et al. Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update. J Clin Oncol. 2013;31(31):3997–4013.CrossRefPubMedGoogle Scholar
  5. 5.
    Sauter G, Lee J, Bartlett JM, Slamon DJ, Press MF. Guidelines for human epidermal growth factor receptor 2 testing: biologic and methodologic considerations. J Clin Oncol. 2009;27(8):1323–33.CrossRefPubMedGoogle Scholar
  6. 6.
    Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SG, Keith DE, et al. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science. 1989;244(4905):707–12.CrossRefPubMedGoogle Scholar
  7. 7.
    Park S, Wang HY, Kim S, Ahn S, Lee D, Cho Y, et al. Quantitative RT-PCR assay of HER2 mRNA expression in formalin-fixed and paraffin-embedded breast cancer tissues. Int J Clin Exp Pathol. 2014;7(10):6752–9.PubMedPubMedCentralGoogle Scholar
  8. 8.
    Perez EA, Cortes J, Gonzalez-Angulo AM, Bartlett JM. HER2 testing: current status and future directions. Cancer Treat Rev. 2014;40(2):276–84.CrossRefPubMedGoogle Scholar
  9. 9.
    Paik S, Shak S, Tang G, Kim C, Baker J, Cronin M, et al. A multigene assay to predict recurrence of tamoxifen-treated, node-negative breast cancer. N Engl J Med. 2004;351(27):2817–26.CrossRefPubMedGoogle Scholar
  10. 10.
    Perez EA, Reinholz MM, Hillman DW, Tenner KS, Schroeder MJ, Davidson NE, et al. HER2 and chromosome 17 effect on patient outcome in the N9831 adjuvant trastuzumab trial. J Clin Oncol. 2010;28(28):4307–15.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    De P, Smith BR, Leyland-Jones B. Human epidermal growth factor receptor 2 testing: where are we? J Clin Oncol. 2010;28(28):4289–92.CrossRefPubMedGoogle Scholar
  12. 12.
    Nitta H, Kelly BD, Padilla M, Wick N, Brunhoeber P, Bai I, et al. A gene-protein assay for human epidermal growth factor receptor 2 (HER2): brightfield tricolor visualization of HER2 protein, the HER2 gene, and chromosome 17 centromere (CEN17) in formalin-fixed, paraffin-embedded breast cancer tissue sections. Diagn Pathol. 2012;7:60.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Nistor A, Watson PH, Pettigrew N, Tabiti K, Dawson A, Myal Y. Real-time PCR complements immunohistochemistry in the determination of HER-2/neu status in breast cancer. BMC Clin Pathol. 2006;6:2.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Kulka J, Tokes AM, Kaposi-Novak P, Udvarhelyi N, Keller A, Schaff Z. Detection of HER-2/neu gene amplification in breast carcinomas using quantitative real-time PCR—a comparison with immunohistochemical and FISH results. Pathol Oncol Res. 2006;12:197–204.CrossRefPubMedGoogle Scholar
  15. 15.
    Murthy SK, Magliocco AM, Demetrick DJ. Copy number analysis of c-erb-B2 (HER-2/neu) and topoisomerase IIalpha genes in breast carcinoma by quantitative real-time polymerase chain reaction using hybridization probes and fluorescence in situ hybridization. Arch Pathol Lab Med. 2005;129(1):39–46.PubMedGoogle Scholar
  16. 16.
    Tvrdik D, Stanek L, Skalova H, Dundr P, Velenska Z, Povysil C. Comparison of the IHC, FISH, SISH and qPCR methods for the molecular diagnosis of breast cancer. Mol Med Rep. 2012;6(2):439–43.PubMedGoogle Scholar
  17. 17.
    Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SG, Keith DE, Levin WJ, Stuart SG, Udove J, Ullrich A. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science. 1989;244:707–12.CrossRefPubMedGoogle Scholar
  18. 18.
    Paik S, Kim C, Jeong J, Geyer CE, Romond EH, Mejia-Mejia O, Mamounas EP, Wickerham D, Costantino JP, Wolmark N. Benefit from adjuvant trastuzumab may not be confined to patients with IHC 3 + and/or FISHpositive tumors: central testing results from NSABP B-31. J Clin Oncol. 2007;25(18S):511.Google Scholar
  19. 19.
    Kaufman PA, Broadwater G, Lezon-Geyda K, Dressler LG, Berry D, Friedman P, Winer EP, Hudis C, Ellis MJ, Seidman AD, Harris LN. Correlation of HER2 and chromosome 17 (ch17) copy number with trastuzumab (T) efficacy in CALGB 9840, paclitaxel (P) with or without T in HER2 + and HER2− metastatic breast cancer (MBC). J Clin Oncol. 1009;2007:25.Google Scholar
  20. 20.
    Perez EA, Reinholz M, Hillman DW, Tenner KS, Schroeder MJ, Davidson NE, Martino S, Sledge GW, Harris LN, Gralow JR, Dueck AC, Ketterling RP, Ingle JN, Lingle WL, Kaufman PA, Visscher DW, Jenkins RB. HER2 and chromosome 17 effect on patient outcome in the N9831 adjuvant trastuzumab trial. J Clin Oncol. 2010;28:4307–15.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    De P, Smith BR, Leyland-Jones B. Human epidermal growth factor receptor 2 testing: where are we? J Clin Oncol. 2010;28:4289–92.CrossRefPubMedGoogle Scholar
  22. 22.
    de Cremoux P, Martin EC, Vincent-Salomon A, Dieras V, Barbaroux C, Liva S, et al. Quantitative PCR analysis of c-erb B-2 (HER2/neu) gene amplification and comparison with p185(HER2/neu) protein expression in breast cancer drill biopsies. Int J Cancer. 1999;83(2):157–61.CrossRefPubMedGoogle Scholar
  23. 23.
    Doval DC, Sharma A, Sinha R, Kumar K, Dewan AK, Chaturvedi H, et al. Immunohistochemical profile of breast cancer patients at a tertiary care hospital in New Delhi, India. Asian Pac J Cancer Prev. 2015;16(12):4959–64.CrossRefPubMedGoogle Scholar
  24. 24.
    Shah MV, Wiktor AE, Meyer RG, Tenner KS, Ballman KV, Green SJ, Sukov WR, Ketterling RP, Perez EA, Jenkins RB. Change in Pattern of HER2 Fluorescent in Situ Hybridization (FISH) results in breast cancers submitted for FISH testing: experience of a reference laboratory using us food and drug administration criteria and American Society of Clinical Oncology and College of American Pathologists Guidelines. J Clin Oncol. 2016;34(29):3502–10.CrossRefPubMedGoogle Scholar
  25. 25.
    Dendukuri N, Khetani K, McIsaac M, Brophy J. Testing for HER2-positive breast cancer: a systematic review and cost-effectiveness analysis. CMAJ. 2007;176(10):1429–34.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Muller BM, Kronenwett R, Hennig G, Euting H, Weber K, Bohmann K, et al. 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. 2011;20(1):1–10.CrossRefPubMedGoogle Scholar
  27. 27.
    Ross JS, Fletcher JA, Bloom KJ, Linette GP, Stec J, Clark E, et al. HER-2/neu testing in breast cancer. Am J Clin Pathol. 2003;120(Suppl):S53–71.PubMedGoogle Scholar
  28. 28.
    Goddard KA, Weinmann S, Richert-Boe K, Chen C, Bulkley J, Wax C. HER2 evaluation and its impact on breast cancer treatment decisions. Public Health Genom. 2012;15(1):1–10.CrossRefGoogle Scholar
  29. 29.
    Khoury T, Sait S, Hwang H, Chandrasekhar R, Wilding G, Tan D, et al. Delay to formalin fixation effect on breast biomarkers. Mod Pathol. 2009;22(11):1457–67.CrossRefPubMedGoogle Scholar
  30. 30.
    Bai Y, Cheng H, Bordeaux J, Neumeister V, Kumar S, Rimm DL, et al. Comparison of HER2 and phospho-HER2 expression between biopsy and resected breast cancer specimens using a quantitative assessment method. PLoS One. 2013;8(11):e79901.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Tapia C, Schraml P, Simon R, Al-Kuraya KS, Maurer R, Mirlacher M, et al. HER2 analysis in breast cancer: reduced immunoreactivity in FISH non-informative cancer biopsies. Int J Oncol. 2004;25(6):1551–7.PubMedGoogle Scholar
  32. 32.
    Egervari K, Szollosi Z, Nemes Z. Tissue microarray technology in breast cancer HER2 diagnostics. Pathol Res Pract. 2007;203(3):169–77.CrossRefPubMedGoogle Scholar
  33. 33.
    Chiu KY, Loke SL, Ho FC. Immunohistochemical demonstration of c-erbB-2 oncoprotein in gastric adenocarcinoma: comparison of cryostat and paraffin wax sections and effect of fixation. J Clin Pathol. 1994;47(2):117–21.CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Penault-Llorca F, Adelaide J, Houvenaeghel G, Hassoun J, Birnbaum D, Jacquemier J. Optimization of immunohistochemical detection of ERBB2 in human breast cancer: impact of fixation. J Pathol. 1994;173(1):65–75.CrossRefPubMedGoogle Scholar
  35. 35.
    Starczynski J, Atkey N, Connelly Y, O’Grady T, Campbell FM, di Palma S, et al. HER2 gene amplification in breast cancer: a rogues’ gallery of challenging diagnostic cases: UKNEQAS interpretation guidelines and research recommendations. Am J Clin Pathol. 2012;137(4):595–605.CrossRefPubMedGoogle Scholar
  36. 36.
    Ginestier C, Charafe-Jauffret E, Penault-Llorca F, Geneix J, Adelaide J, Chaffanet M, Mozziconacci MJ, Hassoun J, Viens P, Birnbaum D, Jacquemier J. Comparative multi methodological measurement of ERBB2 status in breast cancer. J Pathol. 2004;202:286–98.CrossRefPubMedGoogle Scholar
  37. 37.
    Vanden Bempt I, Vanhentenrijk V, Drijkoningen M, Wlodarska I, Vandenberghe P, De Wolf-Peeters C. Real-time reverse transcription-PCR and fluorescence in situ hybridization are complementary to understand the mechanisms involved in HER-2/neu overexpression in human breast carcinomas. Histopathology. 2005;46:431–41.CrossRefPubMedGoogle Scholar
  38. 38.
    Cronin M, Pho M, Dutta D, Stephans JC, Shak S, Kiefer MC, Esteban JM, Baker JB. 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. 2004;164:35–42.CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Vinatzer U, Dampier B, Streubel B, Pacher M, Seewald MJ, Stratowa C, Kaserer K, Schreiber M. Expression of HER2 and the coamplified genes GRB7 and MLN64 in human breast cancer: quantitative real-time reverse transcription-PCR as a diagnostic alternative to immunohistochemistry and fluorescence in situ hybridization. Clin Cancer Res. 2005;11:8348–57.CrossRefPubMedGoogle Scholar
  40. 40.
    Esteva FJ, Sahin AA, Cristofanilli M, Coombes K, Lee SJ, Baker J, Cronin M, Walker M, Watson D, Shak S, Hortobagyi GN. Prognostic role of a multigene reverse transcriptase-PCR assay in patients with node-negative breast cancer not receiving adjuvant systemic therapy. Clin Cancer Res. 2005;11:3315–9.CrossRefPubMedGoogle Scholar
  41. 41.
    Bossard C, Bieche I, Le Doussal V, Lidereau R, Sabourin JC. Real-timebRT-PCR: a complementary method to detect HER-2 status in breast carcinoma. Anticancer Res. 2005;25:4679–83.PubMedGoogle Scholar
  42. 42.
    Kostopoulou E, Vageli D, Kaisaridou D, Lidereau R, Sabourin JC. Comparative evaluation of non-informative HER-2 immunoreactions (2+) in breast carcinomas with FISH, CISH and QRT-PCR. Breast. 2007;16:615–24.CrossRefPubMedGoogle Scholar
  43. 43.
    Barberis M, Pellegrini C, Cannone M, Arizzi C, Coggi G, Bosari S. Quantitative analysis. Am J Clin Pathol. 2008;129:563–70.CrossRefPubMedGoogle Scholar
  44. 44.
    Cuadros M, Talavera P, Lopez FJ, Garcia-Perez I, Blanco A, Concha A. Realtime RT-PCR analysis for evaluating the Her2/neu status in breast cancer. Pathobiology. 2010;77:38–45.CrossRefPubMedGoogle Scholar
  45. 45.
    Zoppoli G, Garuti A, Cirmena G, Cantogno LV, Botta C, Gallo M, Ferraioli D, Carminati E, Baccini P, Curto M, Fregatti P, Isnaldi E, Lia M, Murialdo R, Friedman D, Sapino A. Ballestrero A (2017) Her2 assessment using quantitative reverse transcriptase polymerase chain reaction reliably identifies Her2 overexpression without amplification in breast cancer cases. J Transl Med. 2017;15:91.CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Baehner FL, Achacoso N, Maddala T, Shak S, Quesenberry CP, Goldstein LC, Gown AM, Habel LA. Human epidermal growth factor receptor 2 assessment in a case–control study: comparison of fluorescence in situ hybridization and quantitative reverse transcription polymerase chain reaction performed by central laboratories. J Clin Oncol. 2010;28:4300–6.CrossRefPubMedGoogle Scholar
  47. 47.
    Dabbs DJ, Klein ME, Mohsin SK, Tubbs R, Shuai Y, Bhargava R. High falsenegative rate of HER2 quantitative reverse transcription polymerase chain reaction of the oncotype DX test: an independent quality assurance study. J Clin Oncol. 2011;29:4279–85.CrossRefPubMedGoogle Scholar
  48. 48.
    Lehmann-Che J, Amira-Bouhidel F, Turpin E, Antoine M, Soliman H, Legres L, Bocquet C, Bernoud R, Flandre E, Varna M, de Roquancourt A, Plassa LF, Giacchetti S, Espie M, de Bazelaire C, Cahen-Doidy L, Bourstyn E, Janin A, de The H, Bertheau P. Immunohistochemical and molecular analyses of HER2 status in breast cancers are highly concordant and complementary approaches. Br J Cancer. 2011;104:1739–46.CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    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. 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. 2011;126:109–17.CrossRefPubMedGoogle Scholar
  50. 50.
    Viale G, Slaets L, Bogaerts J, Rutgers E, van’t Veer L, Piccart-Gebhart MJ, de Snoo FA, Stork-Sloots L, Russo L, Dell’Orto P, van den Akker J, Glas A, Cardoso F, Consortium T. High concordance of protein (by IHC), gene (by FISH; HER2 only), and microarray readout (by TargetPrint) of ER, PgR, and HER2: results from the EORTC 10041/BIG 03-04 MINDACT trial. Ann Oncol. 2014;25:816–23.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© The Japanese Breast Cancer Society 2018

Authors and Affiliations

  • Moushumi Suryavanshi
    • 1
  • Anurag Mehta
    • 2
  • Jiten Jaipuria
    • 3
  • Dushyant Kumar
    • 1
  • Gayatri Vishwakarma
    • 4
  • Manoj Kumar Panigrahi
    • 1
  • Haristuti Verma
    • 1
  • Mumtaz Saifi
    • 1
  • Sanjeev Sharma
    • 1
  • Simran Tandon
    • 5
  • D. C. Doval
    • 6
  • Bhudev C. Das
    • 5
  1. 1.Centre for Molecular Diagnostic and Cell BiologyRajiv Gandhi Cancer Institute and Research CentreNew DelhiIndia
  2. 2.Lab Services, Blood Transfusion & MolecularRajiv Gandhi Cancer Institute and research CentreNew DelhiIndia
  3. 3.Department of UrogynaeoncologyRajiv Gandhi Cancer Institute and Research CentreNew DelhiIndia
  4. 4.Department of ResearchRajiv Gandhi Cancer Institute and Research CentreNew DelhiIndia
  5. 5.Amity Institute of Molecular Medicine and Stem Cell Research (AIMMSCR) Amity UniversityNoidaIndia
  6. 6.Medical OncologyRajiv Gandhi Cancer Institute and Research CentreNew DelhiIndia

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