The purpose was to evaluate and compare 5 different HER2 genetic assays with different characteristics that could affect the performance to analyze the human epidermal growth factor 2 (HER2) gene copy number under low and high throughput conditions. The study included 108 tissue samples from breast cancer patients with HER2 immunohistochemistry (IHC) results scored as 0/1+, 2+, and 3+. HER2 genetic status was analysed using chromogenic in situ hybridization (CISH) and fluorescence in situ hybridization (FISH). Scoring results were documented through digital image analysis. The cancer region of interest was identified from a serial H&E stained slide following tissue cores were transferred to a tissue microarrays (TMA). When using TMA in a routine flow, all patients will be tested for HER2 status with IHC followed by CISH or FISH, thereby providing individual HER2 results. In conclusion, our results show that the differences between the HER2 genetic assays do not have an effect on the analytic performance and the CISH technology is superior to high throughput HER2 genetic testing due to scanning speed, while the IQ-FISH may still be a choice for fast low throughput HER2 genetic testing. 1. Introduction Human Epidermal growth factor Receptor 2 (HER2) expression is investigated routinely on all breast cancer cases to make the therapeutic decisions for patients with breast cancer. The American Society of Clinical Oncology (ASCO)/College of American Pathologist (CAP) recommendations for HER2 status testing are first immunohistochemical (IHC) staining and secondary to perform genetic HER2 testing on tissues scored as borderline cases (2+) found by IHC [1]. Ratio-based dual color HER2 gene amplification assays are commercially available from a multiple vendors using either fluorescence in situ hybridization (FISH) or chromogenic in situ hybridization (CISH), where the various tests have differing characteristics (Table 1). The HER2/neu labeled part of the dual color HER2 genetic assays is in all cases DNA based, while the centromere reference part can either be an DNA probe or an peptide nucleic acids (PNA) probe. The direct labeling of the different FISH probes from Dako and ZytoVision uses red (TexasRed), orange (Rhodamine), or green (FITC) fluorocrome, while the CISH-based assays give rise to either red, green, or blue chromogenic precipitation. Various strategies for blocking of nonspecific probe binding and detection systems have been implemented into the different HER2 genetic assays. ZytoVison uses repeat-free oligonucleotides and thereby does not need
References
[1]
A. C. Wolff, M. E. H. Hammond, J. N. Schwartz et al., “American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer,” Archives of Pathology and Laboratory Medicine, vol. 131, no. 1, pp. 18–43, 2007.
[2]
D. Mayr, S. Heim, K. Weyrauch et al., “Chromogenic in situ hybridization for Her-2/neu-oncogene in breast cancer: comparison of a new dual-colour chromogenic in situ hybridization with immunohistochemistry and fluorescence in situ hybridization,” Histopathology, vol. 55, no. 6, pp. 716–723, 2009.
[3]
K. V. Nielsen, S. Müller, T. S. Poulsen, S. Gabs, and A. Schonau, “Chapter 11: combined use of PNA and DNA for fluorescence in situ hybridization (FISH),” in Peptide Nucleic Acids: Protocols and Applications, P. E. Nielsen and M. Egholm, Eds., pp. 227–260, Horizon Bioscience, Norfolk, UK, 2nd edition, 2004.
[4]
C. M. Hansen, “Chapter 14: applications–environmental stress cracking in polymers,” in Hansen Solubility Parameters: A User's Handbook, pp. 269–292, Taylor & Francis, 2nd edition, 2007.
[5]
G. Pauletti, W. Godolphin, M. F. Press, and D. J. Slamon, “Detection and quantitation of HER-2/neu gene amplification in human breast cancer archival material using fluorescence in situ hybridization,” Oncogene, vol. 13, no. 1, pp. 63–72, 1996.
[6]
M. Tanner, D. Gancberg, B. A. D. Leo et al., “Chromogenic in situ hybridization: a practical alternative for fluorescence in situ hybridization to detect HER-2/neu oncogene amplification in archival breast cancer samples,” The American Journal of Pathology, vol. 157, no. 5, pp. 1467–1472, 2000.
[7]
H. H. Rossing, M.-L. M. Talman, A.-V. L？nkholm, and V. T. Wielenga, “Implementation of TMA and digitalization in routine diagnostics of breast pathology,” APMIS, vol. 120, no. 4, pp. 341–347, 2012.
[8]
T. J. A. Dekker, S. T. Borg, G. K. J. Hooijer et al., “Determining sensitivity and specificity of HER2 testing in breast cancer using a tissue micro-array approach,” vol. 120, pp. 341–347, 2012.
[9]
K. Park, J. Kim, S. Lim, S. Han, and J. Y. Lee, “Comparing fluorescence in situ hybridization and chromogenic in situ hybridization methods to determine the HER2/neu status in primary breast carcinoma using tissue microarray,” Modern Pathology, vol. 16, no. 9, pp. 937–943, 2003.
[10]
G. D. Francis, M. A. Jones, G. F. Beadle, and S. R. Stein, “Bright-field in situ hybridization for HER2 gene amplification in breast cancer using tissue microarrays: correlation between chromogenic (CISH) and automated silver-enhanced (SISH) methods with patient outcome,” Diagnostic Molecular Pathology, vol. 18, no. 2, pp. 88–95, 2009.
[11]
A. D. Graham, D. Faratian, F. Rae, and J. S. J. Thomas, “Tissue microarray technology in the routine assessment of HER-2 status in invasive breast cancer: a prospective study of the use of immunohistochemistry and fluorescence in situ hybridization,” Histopathology, vol. 52, no. 7, pp. 847–855, 2008.
[12]
K. E. Olsen, H. Knudsen, B. B. Rasmussen et al., “Amplification of HER2 and TOP2A and deletion of TOP2A genes in breast cancer investigated by new FISH probes,” Acta Oncologica, vol. 43, no. 1, pp. 35–42, 2004.
[13]
Y. Gong, W. Sweet, Y.-J. Duh et al., “Chromogenic in situ hybridization is a reliable method for detecting HER2 gene status in breast cancer a multicenter study using conventional scoring criteria and the new asco/cap recommendations,” American Journal of Clinical Pathology, vol. 131, no. 4, pp. 490–497, 2009.
[14]
J. R. Landis and G. G. Koch, “The measurement of observer agreement for categorical data,” Biometrics, vol. 33, no. 1, pp. 159–174, 1977.
[15]
D. Mayr, S. Heim, K. Weyrauch et al., “Chromogenic in situ hybridization for Her-2/neu-oncogene in breast cancer: comparison of a new dual-colour chromogenic in situ hybridization with immunohistochemistry and fluorescence in situ hybridization,” Histopathology, vol. 55, no. 6, pp. 716–723, 2009.
[16]
T. García-Caballero, D. Grabau, A. R. Green et al., “Determination of HER2 amplification in primary breast cancer using dual-colour chromogenic in situ hybridization is comparable to fluorescence in situ hybridization: a European Multicentre Study involving 168 specimens,” Histopathology, vol. 56, no. 4, pp. 472–480, 2010.
[17]
D. Gupta, L. P. Middleton, M. J. Whitaker, and J. Abrams, “Comparison of fluorescence and chromogenic in situ hybridization for detection of HER-2/neu oncogene in breast cancer,” American Journal of Clinical Pathology, vol. 119, no. 3, pp. 381–387, 2003.
[18]
J. Jacquemier, F. Spyratos, B. Esterni et al., “SISH/CISH or qPCR as alternative techniques to FISH for determination of HER2 amplification status on breast tumors core needle biopsies: a multicenter experience based on 840 cases,” BMC Cancer, vol. 13, pp. 351–361, 2013.
[19]
H. Seol, H. J. Lee, Y. Choi et al., “Intratumoral heterogeneity of HER2 gene amplification in breast cancer: its clinicopathological significance,” Modern Pathology, vol. 25, pp. 938–948, 2012.
[20]
J. Starczynski, N. Atkey, Y. Connelly et al., “HER2 gene amplification in breast cancer: a rogues' gallery of challenging diagnostic cases: UKNEQAS interpretation guidelines and research recommendations,” American Journal of Clinical Pathology, vol. 137, no. 4, pp. 595–605, 2012.
