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Comparative Study
. 2022 Oct;35(10):1362-1369.
doi: 10.1038/s41379-022-01104-9. Epub 2022 Jun 21.

Systematically higher Ki67 scores on core biopsy samples compared to corresponding resection specimen in breast cancer: a multi-operator and multi-institutional study

Balazs Acs  1   2   3 , Samuel C Y Leung  4 , Kelley M Kidwell  5 , Indu Arun  6 , Renaldas Augulis  7 , Sunil S Badve  8 , Yalai Bai  9 , Anita L Bane  10 , John M S Bartlett  11   12 , Jane Bayani  11 , Gilbert Bigras  13 , Annika Blank  14   15 , Henk Buikema  16 , Martin C Chang  17 , Robin L Dietz  18 , Andrew Dodson  19 , Susan Fineberg  20 , Cornelia M Focke  21 , Dongxia Gao  4 , Allen M Gown  22 , Carolina Gutierrez  23 , Johan Hartman  24   25 , Zuzana Kos  26 , Anne-Vibeke Lænkholm  27 , Arvydas Laurinavicius  7 , Richard M Levenson  28 , Rustin Mahboubi-Ardakani  28 , Mauro G Mastropasqua  29 , Sharon Nofech-Mozes  30 , C Kent Osborne  23 , Frédérique M Penault-Llorca  31   32 , Tammy Piper  12 , Mary Anne Quintayo  11 , Tilman T Rau  14   33 , Stefan Reinhard  14 , Stephanie Robertson  24   25 , Roberto Salgado  34   35 , Tomoharu Sugie  36 , Bert van der Vegt  16 , Giuseppe Viale  29   37 , Lila A Zabaglo  38 , Daniel F Hayes  39 , Mitch Dowsett  38 , Torsten O Nielsen  4 , David L Rimm  40 ; International Ki67 in Breast Cancer Working Group of the Breast International Group and North American Breast Cancer Group (BIG-NABCG)
Collaborators, Affiliations
Comparative Study

Systematically higher Ki67 scores on core biopsy samples compared to corresponding resection specimen in breast cancer: a multi-operator and multi-institutional study

Balazs Acs et al. Mod Pathol. 2022 Oct.

Abstract

Ki67 has potential clinical importance in breast cancer but has yet to see broad acceptance due to inter-laboratory variability. Here we tested an open source and calibrated automated digital image analysis (DIA) platform to: (i) investigate the comparability of Ki67 measurement across corresponding core biopsy and resection specimen cases, and (ii) assess section to section differences in Ki67 scoring. Two sets of 60 previously stained slides containing 30 core-cut biopsy and 30 corresponding resection specimens from 30 estrogen receptor-positive breast cancer patients were sent to 17 participating labs for automated assessment of average Ki67 expression. The blocks were centrally cut and immunohistochemically (IHC) stained for Ki67 (MIB-1 antibody). The QuPath platform was used to evaluate tumoral Ki67 expression. Calibration of the DIA method was performed as in published studies. A guideline for building an automated Ki67 scoring algorithm was sent to participating labs. Very high correlation and no systematic error (p = 0.08) was found between consecutive Ki67 IHC sections. Ki67 scores were higher for core biopsy slides compared to paired whole sections from resections (p ≤ 0.001; median difference: 5.31%). The systematic discrepancy between core biopsy and corresponding whole sections was likely due to pre-analytical factors (tissue handling, fixation). Therefore, Ki67 IHC should be tested on core biopsy samples to best reflect the biological status of the tumor.

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Conflict of interest statement

In the last 12 months, DLR has served as a consultant for advisor to Astra Zeneca, Agendia, Amgen, Cell Signaling Technology, Cepheid, Danaher, Konica-Minolta, Merck, PAIGE.AI, Regeneron, and Sanofi. TON reports a proprietary interest in PAM50/Prosigna and consultant work with Veracyte. JH was former member of the advisory board at Visiopharm A/S. JH has obtained speaker’s honoraria or advisory board remunerations from Roche, Novartis, AstraZeneca, Eli Lilly, Pfizer and MSD. JH is co-founder and shareholder of Stratipath AB. JH has received institutional research grants from Cepheid and Novartis. DFH reports no research or personal financial support related to this study. DFH does report research support unrelated to this study provided to his institution during conduct from Menarini/Silicon BioSystems, Astra Zeneca, Eli Lilly Company, Merrimack Pharmaceuticals, Inc. (Parexel Intl Corp), Veridex and Janssen Diagnostics (Johnson & Johnson), Pfizer, and Puma Biotechnology, Inc. (subcontract Wash Univ St. Louis to Univ Mich). DFH also reports that his institution holds a patent regarding circulating tumor cell characterization for which DFH is the named investigator that was licensed to Menarini Silicon Biosystems and from which both received annual royalties, ending in January 2021. DFH reports personal income related to consulting or advisory board activities from BioVeca, Cellworks, Cepheid, EPIC Sciences, Freenome, Guardant, L-Nutra, Oncocyte, Macrogenics, Predictus BioSciences, Salutogenic Innovations, Turnstone Biologics, and Tempus. DFH reports personally held stock options from InBiomotion. BvdV reports speaker’s honoraria or consultation/advisory board remunerations provided to his institution from Visiopharm, Philips, Merck/MSD and Diaceutics. GV reports receipt of grants/research supports from Roche/Genentech, Ventana Medical Systems, Dako/Agilent Technologies, and receipt of honoraria or consultation fees from Ventana, Dako/Agilent, Roche, MSD Oncology, AstraZeneca, Daiichi Sankyo, Pfizer, Eli Lilly. ZK has served in a paid advisory role to Eli Lilly. Unrelated to this study, SR is currently employed by Stratipath AB. RS reports non-financial support from Merck and Bristol Myers Squibb; research support from Merck, Puma Biotechnology, and Roche; and advisory board fees for Bristol Myers Squibb; and personal fees from Roche for an advisory board related to a trial-research project. RS has no COI related to this project. SF served as expert pathology consultant for Axdev Global Corp inc and as an expert advisory panel for Genomic Health in 2017. RML is co-founder and shareholder of MUSE Microscopy, Inc. and Histolix, Inc. RML served as consultant for Cell IDx, Inc., BriteSeed, Inc., ImmunoPhotonics, Inc., Pathology Watch, Inc. and Verily, Inc. FPL reports board meetings and conference support from Astrazeneca, Lilly, Novartis, Pfizer and Roche. JB served as consultant for Insight Genetics, Inc., BioNTech AG, Biotheranostics, Inc., Pfizer, Rna Diagnostics Inc., oncoXchange/MedcomXchange Communications Inc, Herbert Smith French Solicitors, OncoCyte Corporation. JB served as member of the scientific advisory board for MedcomXchange Communications Inc. JB reports honoraria from NanoString Technologies, Inc., Oncology Education, Biotheranostics, Inc., MedcomXchange Communications Inc. JB reports travel and accommodation expenses support from Biotheranostics, Inc., NanoString Technologies, Inc., Breast Cancer Society of Canada. JB received research funding from Thermo Fisher Scientific, Genoptix, Agendia, NanoString Technologies, Inc., Stratifyer GmbH, Biotheranostics, Inc. The remaining authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Study design.
Thirty patients of ER-positive breast cancer were enrolled comprising 15 cases from UK and 15 cases from Japan. Corresponding core-cut biopsy and surgical resection blocks were centrally cut two adjacent sections per case and stained with Ki67. Seventeen pathologists from 15 countries were given 60 slides (30 Core cut biopsy slides and 30 surgical resection specimen slides) of Ki67 to score.
Fig. 2
Fig. 2. Digital Image Analysis.
Representative pictures of digital image analysis (DIA) masks on a resection specimen (A, B) and a core biopsy case (C, D). Blue corresponds to Ki67 negative tumor cells, red indicates Ki67 positive tumor cells, green indicates stromal cells and purple marks immune cells. Black corresponds to necrosis and yellow marks other detections (false cell detections, noise).
Fig. 3
Fig. 3. Between-(consecutive) section difference in Ki67 scoring.
Bland–Altman plot comparing Ki67 scores between consecutive sections (A). Orange dashed line corresponds to the expected mean zero difference between Ki67 scores of the two sections. Red line represents the observed mean difference between Ki67 scores of the two sections, namely the observed bias (red dashed lines are the CI of the observed mean difference). Blue lines illustrate the range of agreement (lower and upper limit of agreement) based on 95% of differences (blue dashed lines are the CI of the limits of agreement). Black line is the fitted regression line to detect potential proportional error (black dashed lines are the CI of the regression line). B represents the scatter plot with fitted regression between the Ki67 scores of the two consecutive sections.
Fig. 4
Fig. 4. Between-specimen (CB vs resection specimen) difference in Ki67 scoring.
Bland–Altman plot comparing Ki67 scores between specimens (A). Orange dashed line corresponds the expected mean zero difference between Ki67 scores of the two sections. Red line represents the observed mean difference between Ki67 scores of the two sections, namely the observed bias (red dashed lines are the CI of the observed mean difference). Blue lines illustrate the range of agreement (lower and upper limit of agreement) based on 95% of differences (blue dashed lines are the CI of the limits of agreement). Black line is the fitted regression line to detect potential proportional error (black dashed lines are the CI of the regression line). B shows the distributions of Ki67 scores of the two specimens. The bottom/top of the boxes represent the first (Q1)/third (Q3) quartiles, the bold line inside the box represents the median and the two bars outside the box represent the lowest/highest datum still within 1.5× the interquartile range (Q3–Q1). C represents the scatter plot with fitted regression between the Ki67 scores of the two specimens.
Fig. 5
Fig. 5. Between-specimen (CB vs resection specimen) difference in Ki67 scoring by case and by origin of the cases.
A represents cases collected in the United Kingdom with representative Ki67 IHC images of corresponding CB and resection specimens. B represents cases collected in Japan with representative Ki67 IHC images of corresponding CB and resection specimens. The bottom/top of the boxes represent the first (Q1)/third (Q3) quartiles, the bold line inside the box represents the median and the two bars outside the box represent the lowest/highest datum still within 1.5× the interquartile range (Q3–Q1). Outliers are represented with circles, extreme outliers with asterisk.
See this image and copyright information in PMC

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