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A novel evaluation method for Ki-67 immunostaining in paraffin-embedded tissues

Virchows Archiv volume 479, pages 121–131 (2021)Cite this article

Abstract

The Ki-67 labeling index is traditionally used to investigate tumor aggressiveness. However, no diagnostic or prognostic value has been associated to the heterogeneous pattern of nuclear positivity. The aims of this study were to develop a classification for the patterns of Ki-67-positive nuclei; to search scientific evidence for the Ki-67 expression and location throughout the cell cycle; and to develop a protocol to apply the classification of patterns of Ki-67-positive nuclei in squamous epithelium with different proliferative activities. Based on empirical observation of paraffin sections submitted to immunohistochemistry for the determination of Ki-67 labeling index and literature review about Ki-67 expression, we created a classification of the patterns of nuclear positivity (NP1, NP2, NP3, NP4, and mitosis). A semi-automatic protocol was developed to identify and quantify the Ki-67 immunostaining patterns in target tissues. Two observers evaluated 7000 nuclei twice to test the intraobserver reliability, and six evaluated 1000 nuclei to the interobserver evaluation. The results showed that the immunohistochemical patterns of Ki-67 are similar in the tumoral and non-tumoral epithelium and were classified without difficulty. There was a high intraobserver reliability (Spearman correlation coefficient > 0.9) and moderate interobserver agreement (k = 0.523). Statistical analysis showed that non-malignant epithelial specimens presented a higher number of NP1 (geographic tongue = 83.8 ± 21.8; no lesion = 107.6 ± 52.7; and mild dysplasia = 86.6 ± 25.8) when compared to carcinoma in Situ (46.8 ± 34.8) and invasive carcinoma (72.6 ± 37.9). The statistical evaluation showed significant difference (p < 0.05). Thus, we propose a new way to evaluate Ki-67, where the pattern of its expression may be associated with the dynamics of the cell cycle. Future proof of this association will validate the use of the classification for its possible impact on cancer prognosis and guidance on personalized therapy.

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Acknowledgments

We thank Antônio Carlos and Aracy Peçanha for the assistance with the pathology archive. We thank Dr. Wilhermo Torres for his advice and encouragement. We thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) for funding. A special thanks to Dr. Nei Soares de Menezes, Pathologist at the Barretos Cancer Hospital Fundação Pio XII, SP, Brazil, for his important collaboration at PHH3-immunostaining.

Availability of data and material

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Funding

This study was funded by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)—Brazil, and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ)—Brazil.

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Affiliations

  1. Unidade Integrada de Patologia Especializada (UniPE), Postgraduate Program in Pathology, Pathological Anatomy Service of Hospital Universitário Antônio Pedro, Universidade Federal Fluminense, Niterói, RJ, 23033-900, Brazil

    Eliane Pedra Dias, Nathália Silva Carlos Oliveira, Amanda Oliveira Serra-Campos, Anna Karoline Fausto da Silva, Licínio Esmeraldo da Silva & Karin Soares Cunha

  2. Department of Pathology, School of Medicine, Universidade Federal Fluminense, Niterói, RJ, Brazil

    Eliane Pedra Dias, Nathália Silva Carlos Oliveira & Karin Soares Cunha

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  1. Eliane Pedra Dias
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  2. Nathália Silva Carlos Oliveira
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  3. Amanda Oliveira Serra-Campos
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  4. Anna Karoline Fausto da Silva
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Contributions

All authors contributed to the study conception and design. E.P.D and N.S.C.O designed the experiments. N.S.C.O. and A.O.S.C. performed the literature review and the experiments. E.P.D; N.S.C.O., A.O.S.C.; A.K.F.S., and K.S.C wrote and reviewed the paper. L.E.S. and N.S.C.O. designed and performed the statistical analysis. All authors had access to the study data and reviewed and approved the final manuscript.

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Correspondence to Eliane Pedra Dias.

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This study strictly followed the tenets of the Declaration of Helsinki and was approved by the Human Ethics and Research Committee of Hospital Universitário Antônio Pedro (HUAP), Universidade Federal Fluminense (# 7660941780000).

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Dias, E.P., Oliveira, N.S.C., Serra-Campos, A.O. et al. A novel evaluation method for Ki-67 immunostaining in paraffin-embedded tissues. Virchows Arch 479, 121–131 (2021). https://doi.org/10.1007/s00428-020-03010-4

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Keywords

  • Ki-67
  • Cell cycle
  • Immunohistochemistry
  • Biomarker