Flow cytometric analysis for Ki67 assessment in formalin-fixed paraffin-embedded breast cancer tissue.
Breast cancer
Flow cytometry
Formalin-fixed paraffin-embedded tissue
Immunohistochemistry
Ki67 labelling index
Journal
BMC biology
ISSN: 1741-7007
Titre abrégé: BMC Biol
Pays: England
ID NLM: 101190720
Informations de publication
Date de publication:
26 Aug 2024
26 Aug 2024
Historique:
received:
20
08
2023
accepted:
13
08
2024
medline:
26
8
2024
pubmed:
26
8
2024
entrez:
25
8
2024
Statut:
epublish
Résumé
Pathologists commonly employ the Ki67 immunohistochemistry labelling index (LI) when deciding appropriate therapeutic strategies for patients with breast cancer. However, despite several attempts at standardizing the Ki67 LI, inter-observer and inter-laboratory bias remain problematic. We developed a flow cytometric assay that employed tissue dissociation, enzymatic treatment and a gating process to analyse Ki67 in formalin-fixed paraffin-embedded (FFPE) breast cancer tissue. We demonstrated that mechanical homogenizations combined with thrombin treatment can be used to recover efficiently intact single-cell nuclei from FFPE breast cancer tissue. Ki67 in the recovered cell nuclei retained reactivity against the MIB-1 antibody, which has been widely used in clinical settings. Additionally, since the method did not alter the nucleoskeletal structure of tissues, the nuclei of cancer cells can be enriched in data analysis based on differences in size and complexity of nuclei of lymphocytes and normal mammary cells. In a clinical study using the developed protocol, Ki67 positivity was correlated with the Ki67 LI obtained by hot spot analysis by a pathologist in Japan (rho = 0.756, P < 0.0001). The number of cancer cell nuclei subjected to the analysis in our assay was more than twice the number routinely checked by pathologists in clinical settings. The findings of this study showed the application of this new flow cytometry method could potentially be used to standardize Ki67 assessments in breast cancer.
Sections du résumé
BACKGROUND
BACKGROUND
Pathologists commonly employ the Ki67 immunohistochemistry labelling index (LI) when deciding appropriate therapeutic strategies for patients with breast cancer. However, despite several attempts at standardizing the Ki67 LI, inter-observer and inter-laboratory bias remain problematic. We developed a flow cytometric assay that employed tissue dissociation, enzymatic treatment and a gating process to analyse Ki67 in formalin-fixed paraffin-embedded (FFPE) breast cancer tissue.
RESULTS
RESULTS
We demonstrated that mechanical homogenizations combined with thrombin treatment can be used to recover efficiently intact single-cell nuclei from FFPE breast cancer tissue. Ki67 in the recovered cell nuclei retained reactivity against the MIB-1 antibody, which has been widely used in clinical settings. Additionally, since the method did not alter the nucleoskeletal structure of tissues, the nuclei of cancer cells can be enriched in data analysis based on differences in size and complexity of nuclei of lymphocytes and normal mammary cells. In a clinical study using the developed protocol, Ki67 positivity was correlated with the Ki67 LI obtained by hot spot analysis by a pathologist in Japan (rho = 0.756, P < 0.0001). The number of cancer cell nuclei subjected to the analysis in our assay was more than twice the number routinely checked by pathologists in clinical settings.
CONCLUSIONS
CONCLUSIONS
The findings of this study showed the application of this new flow cytometry method could potentially be used to standardize Ki67 assessments in breast cancer.
Identifiants
pubmed: 39183273
doi: 10.1186/s12915-024-01980-4
pii: 10.1186/s12915-024-01980-4
doi:
Substances chimiques
Ki-67 Antigen
0
Formaldehyde
1HG84L3525
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
181Informations de copyright
© 2024. The Author(s).
Références
Scholzen T, Gerdes J. The Ki-67 protein: from the known and the unknown. J Cell Physiol. 2000;182(3):311–22.
pubmed: 10653597
doi: 10.1002/(SICI)1097-4652(200003)182:3<311::AID-JCP1>3.0.CO;2-9
Sobecki M, Mrouj K, Colinge J, Gerbe F, Jay P, Krasinska L, Dulic V, Fisher D. Cell-Cycle Regulation Accounts for Variability in Ki-67 Expression Levels. Cancer Res. 2017;77(10):2722–34.
pubmed: 28283655
doi: 10.1158/0008-5472.CAN-16-0707
Cuylen S, Blaukopf C, Politi AZ, Müller-Reichert T, Neumann B, Poser I, Ellenberg J, Hyman AA, Gerlich DW. Ki-67 acts as a biological surfactant to disperse mitotic chromosomes. Nature. 2016;535(7611):308–12.
pubmed: 27362226
pmcid: 4947524
doi: 10.1038/nature18610
Bullwinkel J, Baron-Lühr B, Lüdemann A, Wohlenberg C, Gerdes J, Scholzen T. Ki-67 protein is associated with ribosomal RNA transcription in quiescent and proliferating cells. J Cell Physiol. 2006;206(3):624–35.
pubmed: 16206250
doi: 10.1002/jcp.20494
Davey MG, Hynes SO, Kerin MJ, Miller N, Lowery AJ. Ki-67 as a Prognostic Biomarker in Invasive Breast Cancer. Cancers (Basel). 2021;13(17):4455.
pubmed: 34503265
doi: 10.3390/cancers13174455
Nielsen TO, Leung SCY, Rimm DL, Dodson A, Acs B, Badve S, Denkert C, Ellis MJ, Fineberg S, Flowers M, et al. Assessment of Ki67 in breast cancer: Updated recommendations from the International Ki67 in Breast Cancer Working Group. J Natl Cancer Inst. 2021;113(7):808–19.
pubmed: 33369635
doi: 10.1093/jnci/djaa201
Royce M, Osgood C, Mulkey F, Bloomquist E, Pierce WF, Roy A, et al. FDA Approval Summary: Abemaciclib with endocrine therapy for high-risk early breast cancer. J Clin Oncol. 2022;40(11):1155–62.
pubmed: 35084948
pmcid: 8987222
doi: 10.1200/JCO.21.02742
Focke CM, Bürger H, van Diest PJ, Finsterbusch K, Gläser D, Korsching E, Decker T. Interlaboratory variability of Ki67 staining in breast cancer. Eur J Cancer. 2017;84:219–27.
pubmed: 28829990
doi: 10.1016/j.ejca.2017.07.041
Shi SR, Liu C, Taylor CR. Standardization of immunohistochemistry for formalin-fixed, paraffin-embedded tissue sections based on the antigen-retrieval technique: from experiments to hypothesis. J Histochem Cytochem. 2007;55(2):105–9.
pubmed: 16982846
doi: 10.1369/jhc.6P7080.2006
Gong P, Wang Y, Liu G, Zhang J, Wang Z. New insight into Ki67 expression at the invasive front in breast cancer. PLoS ONE. 2013;8(1):e54912.
pubmed: 23382998
pmcid: 3561452
doi: 10.1371/journal.pone.0054912
Lashen A, Toss MS, Green AR, Mongan NP, Rakha E. Ki67 assessment in invasive luminal breast cancer: a comparative study between different scoring methods. Histopathology. 2022;81(6):786–98.
pubmed: 35997652
pmcid: 9826086
doi: 10.1111/his.14781
Ács B, Kulka J, Kovács KA, Teleki I, Tőkés AM, Meczker Á, Győrffy B, Madaras L, Krenács T, Szász AM. Comparison of 5 Ki-67 antibodies regarding reproducibility and capacity to predict prognosis in breast cancer: does the antibody matter? Hum Pathol. 2017;65:31–40.
pubmed: 28188752
doi: 10.1016/j.humpath.2017.01.011
Lindboe CF, Torp SH. Comparison of Ki-67 equivalent antibodies. J Clin Pathol. 2002;55(6):467–71.
pubmed: 12037032
pmcid: 1769671
doi: 10.1136/jcp.55.6.467
Gerdes J, Schwab U, Lemke H, Stein H. Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation. Int J Cancer. 1983;31(1):13–20.
pubmed: 6339421
doi: 10.1002/ijc.2910310104
Polley MY, Leung SC, McShane LM, Gao D, Hugh JC, Mastropasqua MG, Viale G, Zabaglo LA, Penault-Llorca F, Bartlett JM, et al. An international Ki67 reproducibility study. J Natl Cancer Inst. 2013;105(24):1897–906.
pubmed: 24203987
pmcid: 3888090
doi: 10.1093/jnci/djt306
Polley MY, Leung SC, Gao D, Mastropasqua MG, Zabaglo LA, Bartlett JM, McShane LM, Enos RA, Badve SS, Bane AL, et al. An international study to increase concordance in Ki67 scoring. Mod Pathol. 2015;28(6):778–86.
pubmed: 25698062
doi: 10.1038/modpathol.2015.38
Leung SCY, Nielsen TO, Zabaglo L, Arun I, Badve SS, Bane AL, Bartlett JMS, Borgquist S, Chang MC, Dodson A, et al. Analytical validation of a standardized scoring protocol for Ki67: phase 3 of an international multicenter collaboration. NPJ breast cancer. 2016;2:16014.
pubmed: 28721378
pmcid: 5515324
doi: 10.1038/npjbcancer.2016.14
Leung SCY, Nielsen TO, Zabaglo LA, Arun I, Badve SS, Bane AL, Bartlett JMS, Borgquist S, Chang MC, Dodson A, et al. Analytical validation of a standardised scoring protocol for Ki67 immunohistochemistry on breast cancer excision whole sections: an international multicentre collaboration. Histopathology. 2019;75(2):225–35.
pubmed: 31017314
doi: 10.1111/his.13880
Pons L, Hernández-León L, Altaleb A, Ussene E, Iglesias R, Castillo A, Rodríguez-Martínez P, Castella E, Quiroga V, Felip E, et al. Conventional and digital Ki67 evaluation and their correlation with molecular prognosis and morphological parameters in luminal breast cancer. Sci Rep. 2022;12(1):8176.
pubmed: 35581229
pmcid: 9114341
doi: 10.1038/s41598-022-11411-5
Alataki A, Zabaglo L, Tovey H, Dodson A, Dowsett M. A simple digital image analysis system for automated Ki67 assessment in primary breast cancer. Histopathology. 2021;79(2):200–9.
pubmed: 33590538
doi: 10.1111/his.14355
Bigras G, Dong WF, Canil S, Hugh J, Berendt R, Wood G, Yang H. New robust and reproducible stereological IHC Ki67 breast cancer proliferative assessment to replace traditional biased labeling index. Appl Immunohistochem Mol Morphol. 2017;25(10):687–95.
pubmed: 27093453
doi: 10.1097/PAI.0000000000000371
Li L, Han D, Yu Y, Li J, Liu Y. Artificial intelligence-assisted interpretation of Ki-67 expression and repeatability in breast cancer. Diagn Pathol. 2022;17(1):20.
pubmed: 35094693
pmcid: 8802471
doi: 10.1186/s13000-022-01196-6
Acs B, Pelekanou V, Bai Y, Martinez-Morilla S, Toki M, Leung SCY, Nielsen TO, Rimm DL. Ki67 reproducibility using digital image analysis: an inter-platform and inter-operator study. Lab Invest. 2019;99(1):107–17.
pubmed: 30181553
doi: 10.1038/s41374-018-0123-7
Riva G, Nasillo V, Ottomano AM, Bergonzini G, Paolini A, Forghieri F, Lusenti B, Barozzi P, Lagreca I, Fiorcari S, et al. Multiparametric flow cytometry for MRD monitoring in hematologic malignancies: clinical applications and new challenges. Cancers (Basel). 2021;13(18):4582.
pubmed: 34572809
doi: 10.3390/cancers13184582
Craig FE, Foon KA. Flow cytometric immunophenotyping for hematologic neoplasms. Blood. 2008;111(8):3941–67.
pubmed: 18198345
doi: 10.1182/blood-2007-11-120535
Shi SR, Shi Y, Taylor CR. Antigen retrieval immunohistochemistry: review and future prospects in research and diagnosis over two decades. J Histochem Cytochem. 2011;59(1):13–32.
pubmed: 21339172
pmcid: 3201121
doi: 10.1369/jhc.2010.957191
Leers MP, Theunissen PH, Ramaekers FC, Schutte B. Multi-parameter flow cytometric analysis with detection of the Ki67-Ag in paraffin embedded mammary carcinomas. Cytometry. 1997;27(3):283–9.
pubmed: 9041118
doi: 10.1002/(SICI)1097-0320(19970301)27:3<283::AID-CYTO11>3.0.CO;2-P
Reichard A, Asosingh K. Best practices for preparing a single cell suspension from solid tissues for flow cytometry. Cytometry Part A : the journal of the International Society for Analytical Cytology. 2019;95(2):219–26.
pubmed: 30523671
doi: 10.1002/cyto.a.23690
Weimer S, Oertel K, Fuchsbauer HL. A quenched fluorescent dipeptide for assaying dispase- and thermolysin-like proteases. Anal Biochem. 2006;352(1):110–9.
pubmed: 16564490
doi: 10.1016/j.ab.2006.02.029
Olsen JV, Ong SE, Mann M. Trypsin cleaves exclusively C-terminal to arginine and lysine residues. Mol Cell Proteomics. 2004;3(6):608–14.
pubmed: 15034119
doi: 10.1074/mcp.T400003-MCP200
Sobecki M, Mrouj K, Camasses A, Parisis N, Nicolas E, Llères D, Gerbe F, Prieto S, Krasinska L, David A, et al. The cell proliferation antigen Ki-67 organises heterochromatin. eLife. 2016;5:e13722.
pubmed: 26949251
pmcid: 4841783
doi: 10.7554/eLife.13722
Soliman NA, Yussif SM. Ki-67 as a prognostic marker according to breast cancer molecular subtype. Cancer Biol Med. 2016;13(4):496–504.
pubmed: 28154782
pmcid: 5250608
doi: 10.20892/j.issn.2095-3941.2016.0066
Gusterson BA, Ross DT, Heath VJ, Stein T. Basal cytokeratins and their relationship to the cellular origin and functional classification of breast cancer. Breast Cancer Res. 2005;7(4):143–8.
pubmed: 15987465
pmcid: 1175069
doi: 10.1186/bcr1041
Dum D, Menz A, Völkel C, De Wispelaere N, Hinsch A, Gorbokon N, Lennartz M, Luebke AM, Hube-Magg C, Kluth M, et al. Cytokeratin 7 and cytokeratin 20 expression in cancer: A tissue microarray study on 15,424 cancers. Exp Mol Pathol. 2022;126:104762.
pubmed: 35390310
doi: 10.1016/j.yexmp.2022.104762
Korsching E, Packeisen J, Agelopoulos K, Eisenacher M, Voss R, Isola J, van Diest PJ, Brandt B, Boecker W, Buerger H. Cytogenetic alterations and cytokeratin expression patterns in breast cancer: integrating a new model of breast differentiation into cytogenetic pathways of breast carcinogenesis. Lab Invest. 2002;82(11):1525–33.
pubmed: 12429812
doi: 10.1097/01.LAB.0000038508.86221.B3
Keller CR, Ruud KF, Martinez SR, Li W. Identification of the collagen types essential for mammalian breast acinar structures. Gels (Basel, Switzerland). 2022;8(12):837.
pubmed: 36547361
Mitchell WM, Harrington WF. Purification and properties of clostridiopeptidase B (Clostripain). J Biol Chem. 1968;243(18):4683–92.
pubmed: 4971659
doi: 10.1016/S0021-9258(18)93173-X
Badve S, Vladislav IT, Spaulding B, Strickland A, Hernandez S, Bird-Turner L, Dodson C, Elleby B, Phillips T. EP1: a novel rabbit monoclonal antibody for detection of oestrogen receptor α. J Clin Pathol. 2013;66(12):1051–7.
pubmed: 23894168
doi: 10.1136/jclinpath-2012-201391
Troxell ML, Long T, Hornick JL, Ambaye AB, Jensen KC. Comparison of Estrogen and Progesterone Receptor Antibody Reagents Using Proficiency Testing Data. Arch Pathol Lab Med. 2017;141(10):1402–12.
pubmed: 28714765
doi: 10.5858/arpa.2016-0497-OA
Rajković N, Li X, Plataniotis KN, Kanjer K, Radulovic M, Milošević NT. The pan-cytokeratin staining intensity and fractal computational analysis of breast tumor malignant growth patterns prognosticate the occurrence of distant metastasis. Front Oncol. 2018;8:348.
pubmed: 30214894
pmcid: 6125390
doi: 10.3389/fonc.2018.00348
Brotherick I, Robson CN, Browell DA, Shenfine J, White MD, Cunliffe WJ, Shenton BK, Egan M, Webb LA, Lunt LG, et al. Cytokeratin expression in breast cancer: phenotypic changes associated with disease progression. Cytometry. 1998;32(4):301–8.
pubmed: 9701399
doi: 10.1002/(SICI)1097-0320(19980801)32:4<301::AID-CYTO7>3.0.CO;2-K
Kuburich NA, den Hollander P, Pietz JT, Mani SA. Vimentin and cytokeratin: Good alone, bad together. Semin Cancer Biol. 2022;86(Pt 3):816–26.
pubmed: 34953942
doi: 10.1016/j.semcancer.2021.12.006
Vora HH, Patel NA, Rajvik KN, Mehta SV, Brahmbhatt BV, Shah MJ, Shukla SN, Shah PM. Cytokeratin and vimentin expression in breast cancer. Int J Biol Markers. 2009;24(1):38–46.
pubmed: 19404921
doi: 10.1177/172460080902400106
Wingren S, Guerrieri C, Frånlund B, Stål O. Loss of cytokeratins in breast cancer cells using multiparameter DNA flow cytometry is related to both cellular factors and preparation procedure. Anal Cell Pathol. 1995;9(3):229–33.
pubmed: 8562461
Prokocimer M, Davidovich M, Nissim-Rafinia M, Wiesel-Motiuk N, Bar DZ, Barkan R, Meshorer E, Gruenbaum Y. Nuclear lamins: key regulators of nuclear structure and activities. J Cell Mol Med. 2009;13(6):1059–85.
pubmed: 19210577
pmcid: 4496104
doi: 10.1111/j.1582-4934.2008.00676.x
Stanton SE, Disis ML. Clinical significance of tumor-infiltrating lymphocytes in breast cancer. J Immunother Cancer. 2016;4:59.
pubmed: 27777769
pmcid: 5067916
doi: 10.1186/s40425-016-0165-6
Dayal JH, Sales MJ, Corver WE, Purdie CA, Jordan LB, Quinlan PR, Baker L, ter Haar NT, Pratt NR, Thompson AM. Multiparameter DNA content analysis identifies distinct groups in primary breast cancer. Br J Cancer. 2013;108(4):873–80.
pubmed: 23412097
pmcid: 3590677
doi: 10.1038/bjc.2013.42
Miersch C, Stange K, Röntgen M. Effects of trypsinization and of a combined trypsin, collagenase, and DNase digestion on liberation and in vitro function of satellite cells isolated from juvenile porcine muscles. In Vitro Cell Dev Biol Anim. 2018;54(6):406–12.
pubmed: 29785535
pmcid: 5997727
doi: 10.1007/s11626-018-0263-5
Baxi V, Edwards R, Montalto M, Saha S. Digital pathology and artificial intelligence in translational medicine and clinical practice. Mod Pathol. 2022;35(1):23–32.
pubmed: 34611303
doi: 10.1038/s41379-021-00919-2
Himuro T, Horimoto Y, Arakawa A, Tanabe M, Saito M. Ki67 heterogeneity in estrogen receptor-positive breast cancers: which tumour type has the most heterogeneity? Int J Surg Pathol. 2016;24(2):103–7.
pubmed: 26353854
doi: 10.1177/1066896915605179
Jang MH, Kim HJ, Chung YR, Lee Y, Park SY. A comparison of Ki-67 counting methods in luminal breast cancer: the average method vs. the hot spot method. PLoS One. 2017;12(2):e0172031.
pubmed: 28187177
pmcid: 5302792
doi: 10.1371/journal.pone.0172031
Rakha EA, Reis-Filho JS, Baehner F, Dabbs DJ, Decker T, Eusebi V, Fox SB, Ichihara S, Jacquemier J, Lakhani SR, et al. Breast cancer prognostic classification in the molecular era: the role of histological grade. Breast Cancer Res. 2010;12(4):207.
pubmed: 20804570
pmcid: 2949637
doi: 10.1186/bcr2607
Milbury CA, Creeden J, Yip WK, Smith DL, Pattani V, Maxwell K, Sawchyn B, Gjoerup O, Meng W, Skoletsky J, et al. Clinical and analytical validation of FoundationOne®CDx, a comprehensive genomic profiling assay for solid tumors. PLoS ONE. 2022;17(3):e0264138.
pubmed: 35294956
pmcid: 8926248
doi: 10.1371/journal.pone.0264138
Cardoso F, Kyriakides S, Ohno S, Penault-Llorca F, Poortmans P, Rubio IT, Zackrisson S, Senkus E. Early breast cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up†. Ann Oncol. 2019;30(8):1194–220.
pubmed: 31161190
doi: 10.1093/annonc/mdz173