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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.

Aims: The nuclear proliferation marker Ki67 assayed by immunohistochemistry has multiple potential uses in breast cancer, but an unacceptable level of interlaboratory variability has hampered its clinical utility. The International Ki67 in Breast Cancer Working Group has undertaken a systematic programme to determine whether Ki67 measurement can be analytically validated and standardised among laboratories. This study addresses whether acceptable scoring reproducibility can be achieved on excision whole sections. Methods and results: Adjacent sections from 30 primary ER+ breast cancers were centrally stained for Ki67 and sections were circulated among 23 pathologists in 12 countries. All pathologists scored Ki67 by two methods: (i) global: four fields of 100 tumour cells each were selected to reflect observed heterogeneity in nuclear staining; (ii) hot‐spot: the field with highest apparent Ki67 index was selected and up to 500 cells scored. The intraclass correlation coefficient (ICC) for the global method [confidence interval (CI) = 0.87; 95% CI = 0.799–0.93] marginally met the prespecified success criterion (lower 95% CI ≥ 0.8), while the ICC for the hot‐spot method (0.83; 95% CI = 0.74–0.90) did not. Visually, interobserver concordance in location of selected hot‐spots varies between cases. The median times for scoring were 9 and 6 min for global and hot‐spot methods, respectively. Conclusions: The global scoring method demonstrates adequate reproducibility to warrant next steps towards evaluation for technical and clinical validity in appropriate cohorts of cases. The time taken for scoring by either method is practical using counting software we are making publicly available. Establishment of external quality assessment schemes is likely to improve the reproducibility between laboratories further. [ABSTRACT FROM AUTHOR]

Pathological analysis of the nuclear proliferation biomarker Ki67 has multiple potential roles in breast and other cancers. However, clinical utility of the immunohistochemical (IHC) assay for Ki67 immunohistochemistry has been hampered by unacceptable between-laboratory analytical variability. The International Ki67 Working Group has conducted a series of studies aiming to decrease this variability and improve the evaluation of Ki67. This study tries to assess whether acceptable performance can be achieved on prestained core-cut biopsies using a standardized scoring method. Sections from 30 primary ER+ breast cancer core biopsies were centrally stained for Ki67 and circulated among 22 laboratories in 11 countries. Each laboratory scored Ki67 using three methods: (1) global (4 fields of 100 cells each); (2) weighted global (same as global but weighted by estimated percentages of total area); and (3) hot-spot (single field of 500 cells). The intraclass correlation coefficient (ICC), a measure of interlaboratory agreement, for the unweighted global method (0.87; 95% credible interval (CI): 0.81-0.93) met the prespecified success criterion for scoring reproducibility, whereas that for the weighted global (0.87; 95% CI: 0.7999-0.93) and hot-spot methods (0.84; 95% CI: 0.77-0.92) marginally failed to do so. The unweighted global assessment of Ki67 IHC analysis on core biopsies met the prespecified criterion of success for scoring reproducibility. A few cases still showed large scoring discrepancies. Establishment of external quality assessment schemes is likely to improve the agreement between laboratories further. Additional evaluations are needed to assess staining variability and clinical validity in appropriate cohorts of samples., Competing Interests: J.M.S.B. has consulted for Insight Genetics and BioNTech and received compensation. T.O.N. has consulted for Nanostring and received compensation. C.K.O. has consulted for Astra Zeneca, Genentech and NanoString and received compensation. The remaining authors declare no conflict of interest.