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Free digital image analysis software helps to resolve equivocal scores in HER2 immunohistochemistry

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Abstract

Evaluation of human epidermal growth factor receptor 2 (HER2) immunohistochemistry (IHC) is subject to interobserver variation and lack of reproducibility. Digital image analysis (DIA) has been shown to improve the consistency and accuracy of the evaluation and its use is encouraged in current testing guidelines. We studied whether digital image analysis using a free software application (ImmunoMembrane) can assist in interpreting HER2 IHC in equivocal 2+ cases. We also compared digital photomicrographs with whole-slide images (WSI) as material for ImmunoMembrane DIA. We stained 750 surgical resection specimens of invasive breast cancers immunohistochemically for HER2 and analysed staining with ImmunoMembrane. The ImmunoMembrane DIA scores were compared with the originally responsible pathologists’ visual scores, a researcher’s visual scores and in situ hybridisation (ISH) results. The originally responsible pathologists reported 9.1 % positive 3+ IHC scores, for the researcher this was 8.4 % and for ImmunoMembrane 9.5 %. Equivocal 2+ scores were 34 % for the pathologists, 43.7 % for the researcher and 10.1 % for ImmunoMembrane. Negative 0/1+ scores were 57.6 % for the pathologists, 46.8 % for the researcher and 80.8 % for ImmunoMembrane. There were six false positive cases, which were classified as 3+ by ImmunoMembrane and negative by ISH. Six cases were false negative defined as 0/1+ by IHC and positive by ISH. ImmunoMembrane DIA using digital photomicrographs and WSI showed almost perfect agreement. In conclusion, digital image analysis by ImmunoMembrane can help to resolve a majority of equivocal 2+ cases in HER2 IHC, which reduces the need for ISH testing.

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References

  1. Choritz H, Büsche G, Kreipe H, Study Group HER2 Monitor (2011) Quality assessment of HER2 testing by monitoring of positivity rates. Virchows Arch 459:283–289. doi:10.1007/s00428-011-1132-8

    Article  PubMed  PubMed Central  Google Scholar 

  2. Rydén L, Haglund M, Bendahl PO, et al. (2009) Reproducibility of human epidermal growth factor receptor 2 analysis in primary breast cancer: a national survey performed at pathology departments in Sweden. Acta Oncol 48:860–866. doi:10.1080/02841860902862511

    PubMed  Google Scholar 

  3. Francis GD, Dimech M, Giles L, et al. (2007) Frequency and reliability of oestrogen receptor, progesterone receptor and HER2 in breast carcinoma determined by immunohistochemistry in Australasia: results of the RCPA Quality Assurance Program. J Clin Pathol 60:1277–1283. doi:10.1136/jcp.2006.044701

    Article  PubMed  PubMed Central  Google Scholar 

  4. Vogel UF (2010) Confirmation of a low HER2 positivity rate of breast carcinomas—limitations of immunohistochemistry and in situ hybridization. Diagn Pathol 5:50. doi:10.1186/1746-1596-5-50

    PubMed  PubMed Central  Google Scholar 

  5. Gown AM (2008) Current issues in ER and HER2 testing by IHC in breast cancer. Mod Pathol 21:S8–S15. doi:10.1038/modpathol.2008.34

    Article  CAS  PubMed  Google Scholar 

  6. Saini KS, Azim Jr HA, Metzger-Filho O, et al. (2011) Beyond trastuzumab: new treatment options for HER2-positive breast cancer. Breast 20:S20–S27. doi:10.1016/S0960-9776(11)70289-2

    Article  PubMed  Google Scholar 

  7. Wolff AC, Hammond ME, Schwartz JN, et al. (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 25:118–145. doi:10.1200/JCO.2006.09.2775

    Article  CAS  PubMed  Google Scholar 

  8. Rüschoff J, Hanna W, Bilous M, et al. (2012) HER2 testing in gastric cancer: a practical approach. Mod Pathol 25:637–650. doi:10.1038/modpathol.2011.198

    Article  PubMed  Google Scholar 

  9. Moelans CB, de Weger RA, Van der Wall E, et al. (2011) Current technologies for HER2 testing in breast cancer. Crit Rev Oncol Hematol 80:380–392. doi:10.1016/j.critrevonc.2010.12.005

    Article  CAS  PubMed  Google Scholar 

  10. Wolff AC, Hammond ME, Hicks DG, et al. (2013) 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 31:3997–4013. doi:10.1200/JCO.2013.50.9984

    Article  PubMed  Google Scholar 

  11. Reddy JC, Reimann JD, Anderson SM, et al. (2006) Concordance between central and local laboratory HER2 testing from a community-based clinical study. Clin Breast Cancer 7:153–157. doi:10.3816/CBC.2006.n.025

    Article  PubMed  Google Scholar 

  12. De P, Smith BR, Leyland-Jones B (2010) Human epidermal growth factor receptor 2 testing: where are we? J Clin Oncol 28:4289–4292. doi:10.1200/JCO.2010.29.5071

    Article  PubMed  Google Scholar 

  13. Sauter G, Lee J, Bartlett JM, et al. (2009) Guidelines for human epidermal growth factor receptor 2 testing: biologic and methodologic considerations. J Clin Oncol 27:1323–1333. doi:10.1200/JCO.2007.14.8197

    Article  CAS  PubMed  Google Scholar 

  14. Minot DM, Voss J, Rademacher S, et al. (2012) Image analysis of HER2 immunohistochemical staining. Reproducibility and concordance with fluorescence in situ hybridization of a laboratory-validated scoring technique. Am J Clin Pathol 137:270–276. doi:10.1309/AJCP9MKNLHQNK2ZX

    Article  PubMed  Google Scholar 

  15. Dobson L, Conway C, Hanley A, et al. (2010) Image analysis as an adjunct to manual HER-2 immunohistochemical review: a diagnostic tool to standardize interpretation. Histopathology 57:27–38. doi:10.1111/j.1365-2559.2010.03577.x

    Article  PubMed  PubMed Central  Google Scholar 

  16. Tuominen VJ, Tolonen TT, Isola J (2012) ImmunoMembrane: a publicly available web application for digital image analysis of HER2 immunohistochemistry. Histopathology 60:758–767. doi:10.1111/j.1365-2559.2011.04142.x

    Article  PubMed  Google Scholar 

  17. Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675. doi:10.1038/nmeth.2089

    Article  CAS  PubMed  Google Scholar 

  18. Leong AS, Formby M, Haffajee Z, et al. (2006) Refinement of immunohistologic parameters for Her2/neu scoring validation by FISH and CISH. Appl Immunohistochem Mol Morphol 14:384–389. doi:10.1097/01.pai.0000210415.53493.d4

    Article  CAS  PubMed  Google Scholar 

  19. Hall BH, Ianosi-Irimie M, Javidian P, et al. (2008) Computer-assisted assessment of the human epidermal growth factor receptor 2 immunohistochemical assay in imaged histologic sections using a membrane isolation algorithm and quantitative analysis of positive controls. BMC Med Imaging 8:11. doi:10.1186/1471-2342-8-11

    Article  PubMed  PubMed Central  Google Scholar 

  20. Brügmann A, Eld M, Lelkaitis G, et al. (2012) Digital image analysis of membrane connectivity is a robust measure of HER2 immunostains. Breast Cancer Res Treat 132:41–49. doi:10.1007/s10549-011-1514-2

    Article  PubMed  Google Scholar 

  21. Masmoudi H, Hewitt SM, Petrick N, et al. (2009) Automated quantitative assessment of HER-2/neu immunohistochemical expression in breast cancer. IEEE Trans Med Imaging 28:916–925. doi:10.1109/TMI.2009.2012901

    Article  PubMed  Google Scholar 

  22. Keller B, Chen W, Gavrielides MA (2012) Quantitative assessment and classification of tissue-based biomarker expression with color content analysis. Arch Pathol Lab Med 136:539–550. doi:10.5858/arpa.2011-0195-OA

    Article  PubMed  Google Scholar 

  23. Rojo MG, Bueno G, Slodkowska J (2009) Review of imaging solutions for integrated quantitative immunohistochemistry in the pathology daily practice. Folia Histochem Cytobiol 47:349–354. doi:10.2478/v10042-008-0114-4

    PubMed  Google Scholar 

  24. Rojo MG, García GB, Mateos CP, et al. (2006) Critical comparison of 31 commercially available digital slide systems in pathology. Int J Surg Pathol 14:285–305. doi:10.1177/1066896906292274

    Article  PubMed  Google Scholar 

  25. Nordic Immunohistochemical Quality Control (NordiQC). http://www.nordiqc.org/. Accessed 1 June 2015

  26. Dendukuri N, Khetani K, McIsaac M, et al. (2007) Testing for HER2-positive breast cancer: a systematic review and cost-effectiveness analysis. Cmaj 176:1429–1434. doi:10.1503/cmaj.061011

    Article  PubMed  PubMed Central  Google Scholar 

  27. Cuadros M, Villegas R (2009) Systematic review of HER2 breast cancer testing. Appl Immunohistochem Mol Morphol 17:1–7. doi:10.1097/PAI.0b013e318169fc1c

    Article  CAS  PubMed  Google Scholar 

  28. Minot DM, Kipp BR, Root RM, et al. (2009) Automated cellular imaging system III for assessing HER2 status in breast cancer specimens: development of a standardized scoring method that correlates with FISH. Am J Clin Pathol 132:133–138. doi:10.1309/AJCPJV0SKAF2PCMY

    Article  PubMed  Google Scholar 

  29. Cantaloni C, Tonini RE, Eccher C, et al. (2011) Diagnostic value of automated Her2 evaluation in breast cancer: a study on 272 equivocal (score 2+) Her2 immunoreactive cases using an FDA approved system. Appl Immunohistochem Mol Morphol 19:306–312. doi:10.1097/PAI.0b013e318205b03a

    Article  PubMed  Google Scholar 

  30. Turashvili G, Leung S, Turbin D, et al. (2009) Inter-observer reproducibility of HER2 immunohistochemical assessment and concordance with fluorescent in situ hybridization (FISH): pathologist assessment compared to quantitative image analysis. BMC Cancer 9:165. doi:10.1186/1471-2407-9-165

    Article  PubMed  PubMed Central  Google Scholar 

  31. Laurinaviciene A, Dasevicius D, Ostapenko V, et al. (2011) Membrane connectivity estimated by digital image analysis of HER2 immunohistochemistry is concordant with visual scoring and fluorescence in situ hybridization results: algorithm evaluation on breast cancer tissue microarrays. Diagn Pathol 6:87. doi:10.1186/1746-1596-6-87

    Article  PubMed  PubMed Central  Google Scholar 

  32. Vyberg M, Nielsen S, Røge R, et al. (2015) Immunohistochemical expression of HER2 in breast cancer: socioeconomic impact of inaccurate tests. BMC Health Serv Res 15:352. doi:10.1186/s12913-015-1018-6

    Article  PubMed  PubMed Central  Google Scholar 

  33. Atkinson R, Mollerup J, Laenkholm AV, et al. (2011) Effects of the change in cutoff values for human epidermal growth factor receptor 2 status by immunohistochemistry and fluorescence in situ hybridization: a study comparing conventional brightfield microscopy, image analysis-assisted microscopy, and interobserver variation. Arch Pathol Lab Med 135:1010–1016. doi:10.5858/2010-0462-OAR

    Article  CAS  PubMed  Google Scholar 

  34. Potts SJ, Krueger JS, Landis ND, et al. (2012) Evaluating tumor heterogeneity in immunohistochemistry-stained breast cancer tissue. Lab Investig 92:1342–1357. doi:10.1038/labinvest.2012.91

    Article  PubMed  Google Scholar 

  35. Starczynski J, Atkey N, Connelly Y, et al. (2012) HER2 gene amplification in breast cancer: a rogues’ gallery of challenging diagnostic cases: UKNEQAS interpretation guidelines and research recommendations. Am J Clin Pathol 137:595–605. doi:10.1309/AJCPATBZ2JFN1QQC

    Article  PubMed  Google Scholar 

  36. Walker RA (2006) Quantification of immunohistochemistry—issues concerning methods, utility and semiquantitative assessment I. Histopathology 49:406–410. doi:10.1111/j.1365-2559.2006.02514.x

    Article  CAS  PubMed  Google Scholar 

  37. van der Loos CM (2008) Multiple immunoenzyme staining: methods and visualizations for the observation with spectral imaging. J Histochem Cytochem 56:313–328. doi:10.1369/jhc.2007.950170

    Article  PubMed  PubMed Central  Google Scholar 

  38. Kayser K, Borkenfeld S, Djenouni A et al (2015) Analysis of texture and objects in microscopic images. Diagn Pathol 1:14. doi: 10.17629/www.diagnosticpathology.eu-2015-1:14

  39. Ince DC, Hatton L, Graham-Cumming J (2012) The case for open computer programs. Nature 482:485–488. doi:10.1038/nature10836

    Article  CAS  PubMed  Google Scholar 

  40. Donoho DL (2010) An invitation to reproducible computational research. Biostatistics 11:385–388. doi:10.1093/biostatistics/kxq028

    Article  PubMed  Google Scholar 

  41. Jara-Lazaro AR, Thamboo TP, Teh M, et al. (2010) Digital pathology: exploring its applications in diagnostic surgical pathology practice. Pathology 42:512–518. doi:10.3109/00313025.2010.508787

    Article  PubMed  Google Scholar 

  42. Wilbur DC, Brachtel EF, Gilbertson JR, et al. (2015) Whole slide imaging for human epidermal growth factor receptor 2 immunohistochemistry interpretation: accuracy, precision, and reproducibility studies for digital manual and paired glass slide manual interpretation. J Pathol Inform 6:22. doi:10.4103/2153-3539.157788

    Article  PubMed  PubMed Central  Google Scholar 

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Authors and Affiliations

  1. BioMediTech/Cancer Biology, University of Tampere, 33014, Tampere, Finland

    Henrik O. Helin, Vilppu J. Tuominen, Onni Ylinen & Jorma Isola

  2. HUSLAB, Division of Pathology and Genetics, Helsinki University Central Hospital, P.O. Box 400, 00029 HUS, Finland

    Heikki J. Helin

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  1. Henrik O. Helin
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Helin, H.O., Tuominen, V.J., Ylinen, O. et al. Free digital image analysis software helps to resolve equivocal scores in HER2 immunohistochemistry. Virchows Arch 468, 191–198 (2016). https://doi.org/10.1007/s00428-015-1868-7

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  • DOI: https://doi.org/10.1007/s00428-015-1868-7

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