DOI: | Full Text: [PDF]

Standardization and optimization of fluorescence in situ hybridization (FISH) for HER-2 assessment in breast cancer: A single center experience

Magdalena Bogdanovska-Todorovska, Gordana Petrushevska, Vesna Janevska, Liljana Spasevska, Slavica Kostadinova-Kunovska


Accurate assessment of human epidermal growth factor receptor 2 (HER-2) is crucial in selecting patients for targeted therapy. Commonly used methods for HER-2 testing are immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Here we presented the implementation, optimization and standardization of two FISH protocols using breast cancer samples and assessed the impact of pre-analytical and analytical factors on HER-2 testing. Formalin fixed paraffin embedded (FFPE) tissue samples from 70 breast cancer patients were tested for HER-2 using PathVysion™ HER-2 DNA Probe Kit and two different paraffin pretreatment kits, Vysis/Abbott Paraffin Pretreatment Reagent Kit (40 samples) and DAKO Histology FISH Accessory Kit (30 samples). The concordance between FISH and IHC results was determined. Pre-analytical and analytical factors (i.e., fixation, baking, digestion, and post-hybridization washing) affected the efficiency and quality of hybridization. The overall hybridization success in our study was 98.6% (69/70); the failure rate was 1.4%. The DAKO pretreatment kit was more time-efficient and resulted in more uniform signals that were easier to interpret, compared to the Vysis/Abbott kit. The overall concordance between IHC and FISH was 84.06%, kappa coefficient 0.5976 (p < 0.0001). The greatest discordance (82%) between IHC and FISH was observed in IHC 2+ group. A standardized FISH protocol for HER-2 assessment, with high hybridization efficiency, is necessary due to variability in tissue processing and individual tissue characteristics. Differences in the pre-analytical and analytical steps can affect the hybridization quality and efficiency. The use of DAKO pretreatment kit is time-saving and cost-effective.


Breast cancer; HER-2; fluorescent in situ hybridization; FISH; standardization; IHC; immunohistochemistry; concordance

Full Text:



Hicks DG, Tubbs RR. Assessment of the HER2 status in breast cancer by fluorescence in situ hybridization: A technical review with interpretive guidelines. Hum Pathol 2005;36(3):250-61.

Ross JS, Slodkowska EA, Symmans WF, Pusztai L, Ravdin PM, Hortobagyi GN. The HER-2 receptor and breast cancer: Ten years of targeted anti-HER-2 therapy and personalized medicine. Oncologist 2009;14(4):320-68.

Wolff AC, Hammond ME, Schwartz JN, Hagerty KL, Allred DC, Richard J, et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. Arch Pathol Lab Med 2007;131(1):18-43.

DOI: 10.1043/1543-2165(2007)131[18:ASOCCO]2.0.CO;2.

Stocker A, Hilbers ML, Gauthier C, Grogg J, Kullak-Ublick GA, Seifert B, et al. HER2/CEP17 ratios and clinical outcome in HER2-positive early breast cancer undergoing trastuzumab-containing therapy. PLoS One 2016;11(7):e0159176.

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.

Sapino A, Goia M, Recupero D, Marchiò C. Current challenges for HER2 testing in diagnostic pathology: State of the art and controversial issues. Front Oncol 2013;3:129.

Lottner C, Schwarz S, Diermeier S, Hartmann A, Knuechel R, Hofstaedter F, et al. Simultaneous detection of HER2/neu gene amplification and protein overexpression in paraffin-embedded breast cancer. J Pathol 2005;205(5):577-84.

Dowsett M, Hanna WM, Kockx M, Penault-Llorca F, Ruschoff J, Gutjahr T, et al. Standardization of HER2 testing: Results of an international proficiency-testing ring study. Mod Pathol 2007;20(5):584-91.

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.

Andersen CL, Hostetter G, Grigoryan A, Sauter G, Kallioniemi A. Improved procedure for fluorescence in situ hybridization on tissue microarrays. Cytometry 2001;45(2):83-6.<83::AID-CYTO1149>3.0.CO;2-P.

Petersen BL, Sorensen MC, Pedersen S, Rasmussen M. Fluorescence in situ hybridization on formalin-fixed and paraffin-embedded tissue: Optimizing the method. Appl Immunohistochem Mol Morphol 2004;12(3):259-65.

Pauletti G, Dandekar S, Rong H, Ramos L, Peng H, Seshadri R, et al. Assessment of methods for tissue-based detection of the HER-2/neu alteration in human breast cancer: A direct comparison of fluorescence in situ hybridization and immunohistochemistry. J Clin Oncol 2000;18(21):3651-64.

Gokhale S, Gatalica Z, Mohammad A, Rampy AI, Velagaleti Gopalrao VN. FISH for HER-2/neu in breast cancer: Standardization makes the difference! Indian J Cancer 2004;41(4):152-8.

Mass RD, Press MF, Anderson S, Cobleigh MA, Vogel CL, Dybdal N, et al. Evaluation of clinical outcomes according to HER2 detection by fluorescence in situ hybridization in women with metastatic breast cancer treated with trastuzumab. Clin Breast Cancer 2005;6(3):240-6.

Mascarello JT, Hirsch B, Kearney HM, Ketterling RP, Olson SB, Quigley DI, et al. Section E9 of the American College of Medical Genetics technical standards and guidelines: Fluorescence in situ hybridization. Genet Med 2011;13(7):667-75.

Walker RA, Bartlett JM, Dowsett M, Ellis IO, Hanby AM, Jasani B, et al. HER2 testing in the UK: Further update to recommendations. J Clin Pathol 2008;61(7):818-24.

Perez EA, Cortes J, Gonzalez-Angulo AM, Bartlett JM. HER2 testing: Current status and future directions. Cancer Treat Rev 2014;40(2):276-84.

Hyytinen E, Visakorpi T, Kallioniemi A, Kallioniemi OP, Isola JJ. Improved technique for analysis of formalin-fixed, paraffin-embedded tumors by fluorescence in situ hybridization. Cytometry 1994;16(2):93-9.

Bartlett JM, Starczynski J, Atkey N, Kay E, O’Grady A, Gandy M, et al. HER2 testing in the UK: Recommendations methods for breast and gastric in-situ hybridization. J Clin Pathol 2011;64(8):649-53.

Yaziji H, Goldstein LC, Barry TS, Werling R, Hwang H, Ellis GK, et al. HER-2 testing in breast cancer using parallel tissue-based methods. JAMA 2004;291(16):1972-7.

Provenzano E, Johnson N. Overview of recommendations of HER2 testing in breast cancer. Diagn Histopathol 2009;15(10):478-84.

Sui W, Ou M, Chen J, Wan Y, Peng H, Qi M, et al. Comparison of immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) assessment for her-2 status in breast cancer. World J Surg Oncol 2009;7:83.

Dybdal N, Leiberman G, Anderson S, McCune B, Bajamonde A, Cohen RL, et al. Determination of HER2 gene amplification by fluorescence in situ hybridization and concordance with the clinical trials immunohistochemical assay in women with metastatic breast cancer evaluated for treatment with trastuzumab. Breast Cancer Res Treat 2005;93(1):3-11.

Sarode VR, Xiang QD, Christie A, Collins R, Rao R, Leitch AM, et al. Evaluation of HER2/neu status by immunohistochemistry using computer-based image analysis and correlation with gene amplification by fluorescence in situ hybridization assay: A 10-year experience and impact of test standardization on concordance rate. Arch Pathol Lab Med 2015;139(7):922-8.


  • There are currently no refbacks.

Quick links for Authors:Quick links for Peer Reviewers:

Advanced online | Current Issue | Archives | Submit a manuscript | About the Journal

© Association of Basic Medical Sciences of FBIH, 1998-2018. Credits to Public Knowledge Project