An international Ki67 reproducibility study.

BACKGROUND In breast cancer, immunohistochemical assessment of proliferation using the marker Ki67 has potential use in both research and clinical management. However, lack of consistency across laboratories has limited Ki67's value. A working group was assembled to devise a strategy to harmonize Ki67 analysis and increase scoring concordance. Toward that goal, we conducted a Ki67 reproducibility study. METHODS Eight laboratories received 100 breast cancer cases arranged into 1-mm core tissue microarrays-one set stained by the participating laboratory and one set stained by the central laboratory, both using antibody MIB-1. Each laboratory scored Ki67 as percentage of positively stained invasive tumor cells using its own method. Six laboratories repeated scoring of 50 locally stained cases on 3 different days. Sources of variation were analyzed using random effects models with log2-transformed measurements. Reproducibility was quantified by intraclass correlation coefficient (ICC), and the approximate two-sided 95% confidence intervals (CIs) for the true intraclass correlation coefficients in these experiments were provided. RESULTS Intralaboratory reproducibility was high (ICC = 0.94; 95% CI = 0.93 to 0.97). Interlaboratory reproducibility was only moderate (central staining: ICC = 0.71, 95% CI = 0.47 to 0.78; local staining: ICC = 0.59, 95% CI = 0.37 to 0.68). Geometric mean of Ki67 values for each laboratory across the 100 cases ranged 7.1% to 23.9% with central staining and 6.1% to 30.1% with local staining. Factors contributing to interlaboratory discordance included tumor region selection, counting method, and subjective assessment of staining positivity. Formal counting methods gave more consistent results than visual estimation. CONCLUSIONS Substantial variability in Ki67 scoring was observed among some of the world's most experienced laboratories. Ki67 values and cutoffs for clinical decision-making cannot be transferred between laboratories without standardizing scoring methodology because analytical validity is limited.

[1]  N. Horton Multilevel and Longitudinal Modeling Using Stata , 2006 .

[2]  Arvydas Laurinavicius,et al.  Digital Image Analysis in Pathology: Benefits and Obligation , 2011, Analytical cellular pathology.

[3]  M. Dowsett,et al.  Biomarker changes during neoadjuvant anastrozole, tamoxifen, or the combination: influence of hormonal status and HER-2 in breast cancer--a study from the IMPACT trialists. , 2005, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[4]  R. Bast,et al.  American Society of Clinical Oncology 2007 update of recommendations for the use of tumor markers in breast cancer. , 2007, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[5]  Jarrod D. Hadfield,et al.  MCMC methods for multi-response generalized linear mixed models , 2010 .

[6]  C Caldas,et al.  Proliferation markers and survival in early breast cancer: a systematic review and meta-analysis of 85 studies in 32,825 patients. , 2008, Breast.

[7]  Joseph C Cappelleri,et al.  A modified large-sample approach to approximate interval estimation for a particular intraclass correlation coefficient. , 2003, Statistics in medicine.

[8]  B. Ljung,et al.  Invasive breast cancer. , 2007, Journal of the National Comprehensive Cancer Network : JNCCN.

[9]  S. Kunte,et al.  Statistical computing , 1999 .

[10]  Laura H. Tang,et al.  Objective Quantification of the Ki67 Proliferative Index in Neuroendocrine Tumors of the Gastroenteropancreatic System: A Comparison of Digital Image Analysis With Manual Methods , 2012, The American journal of surgical pathology.

[11]  R Core Team,et al.  R: A language and environment for statistical computing. , 2014 .

[12]  A. Beckett,et al.  AKUFO AND IBARAPA. , 1965, Lancet.

[13]  A. Giobbie-Hurder,et al.  Prognostic and predictive value of centrally reviewed Ki-67 labeling index in postmenopausal women with endocrine-responsive breast cancer: results from Breast International Group Trial 1-98 comparing adjuvant tamoxifen with letrozole. , 2008, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[14]  Soonmyung Paik,et al.  Use of archived specimens in evaluation of prognostic and predictive biomarkers. , 2009, Journal of the National Cancer Institute.

[15]  Mikael Lundin,et al.  Development and evaluation of a virtual microscopy application for automated assessment of Ki-67 expression in breast cancer , 2011, BMC clinical pathology.

[16]  Jack Cuzick,et al.  Assessment of Ki67 in breast cancer: recommendations from the International Ki67 in Breast Cancer working group. , 2011, Journal of the National Cancer Institute.

[17]  Ingvar Andersson,et al.  Invasive breast cancer. , 2011 .

[18]  M. Dowsett,et al.  A randomised study of the effects of letrozole and anastrozole on oestrogen receptor positive breast cancers in postmenopausal women , 2009, Breast Cancer Research and Treatment.

[19]  Vilppu J. Tuominen,et al.  Grading of Neuroendocrine Tumors With Ki-67 Requires High-quality Assessment Practices , 2012, The American journal of surgical pathology.

[20]  T. Kirkegaard,et al.  Observer variation in immunohistochemical analysis of protein expression, time for a change? , 2006, Histopathology.

[21]  Harald Frick,et al.  How Reliable Is Ki-67 Immunohistochemistry in Grade 2 Breast Carcinomas? A QA Study of the Swiss Working Group of Breast- and Gynecopathologists , 2012, PloS one.

[22]  M. Dowsett,et al.  A randomized trial exploring the biomarker effects of neoadjuvant sequential treatment with exemestane and anastrozole in post-menopausal women with hormone receptor-positive breast cancer , 2009, Breast Cancer Research and Treatment.

[23]  M. Dowsett,et al.  A phase II placebo-controlled trial of neoadjuvant anastrozole alone or with gefitinib in early breast cancer. , 2007, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[24]  J. Olson,et al.  Randomized phase II neoadjuvant comparison between letrozole, anastrozole, and exemestane for postmenopausal women with estrogen receptor-rich stage 2 to 3 breast cancer: clinical and biomarker outcomes and predictive value of the baseline PAM50-based intrinsic subtype--ACOSOG Z1031. , 2011, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[25]  Anais Malpica,et al.  Comparison of the effect of different techniques for measurement of Ki67 proliferation on reproducibility and prognosis prediction accuracy in breast cancer , 2012, Histopathology.

[26]  J M Bland,et al.  Statistical methods for assessing agreement between two methods of clinical measurement , 1986 .

[27]  K. Gelmon,et al.  Ki67 in breast cancer: prognostic and predictive potential. , 2010, The Lancet. Oncology.

[28]  Andrzej T. Galecki,et al.  Linear mixed-effects models using R , 2013 .

[29]  Gao Hai-feng,et al.  Proliferation marker Ki-67 in early breast cancer , 2009 .

[30]  S. Teutsch,et al.  The Evaluation of Genomic Applications in Practice and Prevention (EGAPP) initiative: methods of the EGAPP Working Group , 2009, Genetics in Medicine.

[31]  V. Adsay Ki67 Labeling Index in Neuroendocrine Tumors of the Gastrointestinal and Pancreatobiliary Tract: To Count or Not to Count Is Not the Question, But Rather How to Count , 2012, The American journal of surgical pathology.

[32]  E Leonardi,et al.  Proliferative activity in human breast cancer: Ki-67 automated evaluation and the influence of different Ki-67 equivalent antibodies , 2011, Diagnostic pathology.

[33]  I. Ellis,et al.  The Nottingham prognostic index in primary breast cancer , 2005, Breast Cancer Research and Treatment.

[34]  R. Gelber,et al.  Strategies for subtypes—dealing with the diversity of breast cancer: highlights of the St Gallen International Expert Consensus on the Primary Therapy of Early Breast Cancer 2011 , 2011, Annals of oncology : official journal of the European Society for Medical Oncology.