https://openworks.mdanderson.org/sumexp22/1094/thumbnail.jp

Albarracin, Constance

Chen, Hui

Mersereau, Mary

English

OpenWorks @ MD Anderson

Title of the Poster Presentation Goes HereAuthors of the Poster Presentation Goes HereInstitutional and/or Graduate School of Biomedical Sciences Affiliation Goes Here Ki67 is an important tumor marker in breast cancer that isassociated with cell proliferation. Ki67 has been suggested to beof clinical value in determining whether adjuvant chemotherapyis necessary in ER positive and HER2 negative breast cancerpatients. While immunohistochemical stains are commonly usedto assess Ki67, quantitation is limited by lack of standard scoringmethodsMost commonly, the total percentage of Ki67 in the entiretumor is visually evaluated. More recently, the International Ki67Working Group (IKWG) proposed counting 100 cells across fourareas of varying Ki67 density and taking the average of thecounts. Another method is selecting representative areas of“hotspots,” areas with the highest Ki67 nuclear labeling. At thistime however, no single method has gained universalacceptance and a reliable and reproducible method for Ki67quantitation remains to be identified.The goal of this study was to compare and evaluate fourdifferent methods of estimating Ki67 nuclear staining by VisualTotal Count, Digital Total Count, IKWG Unweighted and HotspotCount.Selection of Cases100 cases were chosen from a database of 657 ER positive andHER2 negative invasive breast carcinomas. A consensus reviewof the Ki67 by a team of pathologists was estimated using theVisual Total Count method. The 100 cases were classified intothe following Ki67 groups: 49 Low, 29 Moderate, and 22 Highcases.Immunohistochemistry Paraffin-embedded formalin-fixed slides were incubated with MIB-1/Ki67 (DAKO) antibodies.Analysis of Immunohistochemical Staining Stained slides were digitized using Aperio® AT2 (Leica Biosystems) apparatus. Ki67 immunohistochemical staining were classified as follows: • Low positive expression, <17%• Moderate positive expression, 17%-35%• High positive expression, >35% Table 1: Comparison of Ki67 Visual Total Count to the three different quantitation methods: Digital Total Count, IKWG Unweighted, and Hotspot Count.4) Hotspot CountOur study successfully demonstrated that Visual Total Count, Digital Total Count and IKWG Unweighted are reliable and reproducible methods for Ki67 evaluation. In contrast, the Hotspot Count had a lower correlation with Visual Total Count, and its use in clinical practice may be limited. Further studies to establish the clinical significance of these Ki67 categories and their impact on clinical management are necessary.Ki67 Quantitation in Breast Cancer: A Comparative Analysis of Four Counting MethodologiesINTRODUCTIONMATERIALSCONCLUSIONSFig. 1: Examples of Ki67 staining. IHC stain showing low positive (A), moderate positive (B) and high positive (C) Ki67 staining.Mary Mersereau1,2, Hui Chen MD, PhD1, Constance Albarracin MD, PhD11Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston TX; 2Baylor UniversityTotal percentage of Ki67 nuclear staining within the entire invasive tumor was visually estimated. This method was used as the “gold-standard” method to which all others were compared.2) Digital Total Count A digital image of the slide was obtained. Screenshots of the entire tumor were taken at 10x magnification.  The Ki67 staining of the entire tumor was quantified using digital analysis (Image J).The pathologists selected areas with high, moderate, low, and negligible Ki67 staining within each sample. A digital image was obtained and Ki67 of 100 cells in each area were counted using the manual cell counter function in Aperio®.1) Visual Total Count 3) IKWG UnweightedA screenshot of the most prominent Ki67 labeling was taken at 10x magnification, a color photo was printed and then used to count Ki67 labeling in 400 cells.METHODSFig. 2: Correlation of Ki67 Scoring Between Visual Total Count and the 3 Methods. Scatterplot matrix plots for correlation between Visual Total Count and Digital Total Count (A), IKWG Unweighted (B), and Hotspot Count (C). Line graphs showing relationship between Visual Total Count and Digital Total Count (D), IKWG Unweighted (E), and Hotspot Count (F). • Digital Total Count and IKWG Unweighted yield comparable results to pathologist estimations (Visual Total Count). However, careful selections of intact images for digitized analysis are optimal. • Hotspot Count had a low correlation with Visual Total Count. The error was likely caused by variation in staining throughout samples, making the hotspot a poor representation of the overall Ki67 percentage.The Digital Total Count and IKWG Unweighted methods altered Ki67 categories in 3 cases each.Digital Total Count:• 2 Moderate (20%, 17%) became Low (15.6%,15.7%)• 1 High (35%) became Moderate (32.8%)IKWG Unweighted:• 2 Moderate (20%, 17%) became Low (16.25%, 16%)• 1 Low (15%) became Moderate (19.25%)The Hotspot Count method altered Ki67 categories in 16 cases.REFERENCES1.Owens R, Gilmore E, Bingham V, Cardwell C, McBride H, McQuaid S, Humphries M, Kelly P. Comparison of different anti-Ki67 antibody clones and hot-spot sizes for assessing proliferative index and grading in pancreatic neuroendocrine tumours using manual and image analysis. Histopathology. 2020 Oct;77(4):646-658. doi: 10.1111/his.14200. Epub 2020 Sep 2. PMID: 32617996.2.Albarracin, C., Dhamne, S. Ki67 as a Biomarker of Prognosis and Prediction: Is it Ready for Use in Routine Pathology Practice?. Curr Breast Cancer Rep 6, 260–266 (2014). https://doi.org/10.1007/s12609-014-0163-y3.Reid MD, Bagci P, Ohike N, Saka B, Erbarut Seven I, Dursun N, Balci S, Gucer H, Jang KT, Tajiri T, Basturk O, Kong SY, Goodman M, Akkas G, Adsay V. Calculation of the Ki67 index in pancreatic neuroendocrine tumors: a comparative analysis of four counting methodologies. Mod Pathol. 2015 May;28(5):686-94. doi:4.Nielsen TO, Leung SCY, Rimm DL, Dodson A, Acs B, Badve S, Denkert C, Ellis MJ, Fineberg S, Flowers M, Kreipe HH, Laenkholm AV, Pan H, Penault-Llorca FM, Polley MY, Salgado R, Smith IE, Sugie T, Bartlett JMS, McShane LM, Dowsett M, Hayes DF. Assessment of Ki67 in Breast Cancer: Updated Recommendations from the International Ki67 in Breast Cancer Working Group. J Natl Cancer Inst. 2021 Jul 1;113(7):808-819. doi: 10.1093/jnci/djaa201. PMID: 33369635; PMCID: PMC8487652. 10.1038/modpathol.2014.156. Epub 2014 Nov 21. Erratum in: Mod Pathol. 2016 Jan;29(1):93. PMID: 25412850; PMCID: PMC4460192.A BC00.20.40.60.811 10 19 28 37 46 55 64 73 82 91 100Ki67 (%)Case NumberVisual Total CountHot Spot00.20.40.60.811 10 19 28 37 46 55 64 73 82 91 100Ki67 (%)Case Number Visual Total CountIKWG UnweightedRESULTS00.20.40.60.811 10 19 28 37 46 55 64 73 82 91 100Ki67 (%)Case NumberTotal Count (Visual)Digital Total Count0.00.20.40.60.81.00 0.2 0.4 0.6 0.8 1Digital Total Count (%)Visual Total Count (%)BA C00.20.40.60.810 0.2 0.4 0.6 0.8 1IKWG Unweighted (%)Visual Total Count (%)IKWG Unweighted00.20.40.60.810 0.2 0.4 0.6 0.8 1Hotspot Count (%)Visual Total Count (%)R^2 = 0.8678Hotspot Count EDIKWG Unweighted Hotspot Count Digital Total Count Digital Total Count FR^2 = 0.9776R^2 = 0.9756Visual Total CountKi67 Low (n = 49)Ki67 Moderate (n = 29)Ki67 High (n = 22)Digital Total Count Low (n = 51) 49 2 0Moderate (n = 28) 0 27 1High (n = 21) 0 0 21IKWG Unweighted Low (n =50) 48 2 0Moderate (n = 28) 1 27 0High (n = 22) 0 0 22Hotspot Count Low (n = 42) 40 2 0Moderate (n = 31) 9 21 1High (n = 27) 0 6 211.0 1.01.01.01.01.01.01.0

Ki67 Quantitation in Breast Cancer: A Comparative Analysis of Four Counting Methodologies

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Ki67 Quantitation in Breast Cancer: A Comparative Analysis of Four Counting Methodologies

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