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Abstract Background and Aims Ki-67 is a well-established biomarker of proliferation in breast cancer (BC). However, its value in treatment decision making is hampered by a lack of analytical reproducibility. Regular participation in external quality assessment (EQA) substantially improves inter-laboratory concordance. Pre-requisites in establishing a fit-for-purpose EQA for Ki-67 are a well-validated testing substrate and a reproducible method of assessing Ki-67 scores in participants’ submitted materials. We report here on the results of work using cell line controls analysed by digital image analysis (DIA) as a first step towards providing such an EQA. Materials and Methods A formalin-fixed paraffin embedded (FFPE) cell line microarray (CLMA) was designed and produced in conjunction with Array Sciences LLC (Sausalito, USA). It was comprised of cores taken from a pure population of Sf9 caterpillar cells, which have been shown to be completely unreactive with most commercial antibodies to human Ki-67, together with cores of Sf9 cells mixed with four different human BC cell lines. These were BT-20 (85% BC cells), ZR-75-1 (75%), BT-474 (65%) and BT-483 (55%). Sections from the CLMA were mounted onto glass microscope slides together with sections from a FFPE tonsil sample and two BC samples; one BC showed high (~30%, BC-high) and the second, low proliferation (~5%, BC-low). In both BC samples proliferating cancer cells were distributed homogeneously throughout the the block. Unstained sections were distributed to laboratories participating in the Scheme’s Ki-67 BC programme; after routine IHC-staining for Ki-67 returned slides were centrally visually assessed for stain quality and subjected to DIA using an in-house application developed on Visiopharm software (Visiopharm A/S, Hoersholm, Denmark). Results Slides were returned by 37 laboratories. Analysis of Ki-67 scores obtained on the Sf9 core identified two distinct groups. The first (n = 27) were negative or showed low Ki-67 scores (mean = 1.1%, 95% CIs: 0.2-1.9%), the second (n = 10) displayed a step-change in scores (mean = 49.9%, 95% CIs: 33.2-66.7%); the means of the two groups were significantly different (P 0.0001). When Ki-67 scores for each of the tissue samples were dichotomized into similar groups, the means of those groups differed significantly for the two BC samples (P 0.001), but not for tonsil. Quality scores generated by visual assessment did not differ significantly between the two groups. However, when slides bearing Sf9 cores demonstrating aberrant Ki-67 scores were visually examined nuclear staining was clearly visible, and non-specific nuclear staining could also be identified in the matched BC tissue samples, but not in the tonsil sections. Correlation of Ki-67 scores between the four BC cell line cores and each of the BC tissue samples was examined using Pearson’s correlation statistics. The r statistic range was 0.57-0.71 in comparisons between BC cell line cores and the BC-high sample, and 0.53-0.70 for those with BC-low; in each case BT-483 showed the highest correlation score and BT-20 the lowest. A similar analysis was undertaken between the tonsil and the two BC tissues. The r statistic for correlation between tonsil and BC-high scores was 0.72; it was 0.64 for those between tonsil and BC-low. Conclusions By using a pure population of Sf9 cells we have developed a sensitive indicator of non-specific nuclear staining in IHC preparations stained for Ki-67 which identifies the presence of the artefact quantifiably. Cores made from Sf9/BC cell line mixtures (especially BT-483) produce Ki-67 scores which correlate with those obtained in breast cancer samples at a similar level to those achieved between tonsil and BC samples; this is true for both high and the low proliferation ranges. Cell line mixtures can be adjusted to show Ki-67 scores in the clinically relevant ranges, and they do not show the inherent biological variations seen in tissue controls such as tonsil. Citation Format: Andrew Dodson, Fitim Berisha, Dawn Wilkingson, Lila Zabaglo, Suzanne Parry. Digital image analysis and a novel set of cell line samples as aids in the development of a quantitative external quality assessment programme for Ki-67 abstract. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-26-09.
Dodson et al. (Thu,) studied this question.