Abstract P6-04-09: Variations in diagnostic practice of Ki67 scoring suggest standard guidelines needed for pathological assessment: survey results of global Ki67 testing methods
Authors: Moore Heather M. , Lin Wendy W. , Fernando Tharu M. , Lopez Celine , Kent Emma , Ellouk Karine , Giltnane Jennifer M. 
Published: March 2023

Abstract
BACKGROUND The cell proliferation biomarker Ki67 is expressed during every phase of the cell cycle and has long been used as a diagnostic tool for cancer prognosis, especially for hormone receptor positive breast cancer (HR+ BC). More recently, Ki67 has emerged as a companion diagnostic to select patients for the medicines targeting high risk HR+ BC. We report survey results of local Ki67 immunohistochemistry (IHC) testing practices across 99 pathology labs in 11 countries supporting clinical trial sites in 2020-2021 for the coopERA Breast Cancer clinical study in neoadjuvant HR+ BC (NCT04436744). METHODS The survey was disseminated to pathology labs across five continents to assess local Ki67 IHC staining, analysis, and scoring methodologies. Metrics included pre-analytical considerations (e.g. sample type and requirements) and analytical considerations (e.g. test validation status, antibody, scoring methods, reporting). RESULTS All pathology labs reported requiring formalin-fixed, paraffin-embedded (FFPE) tissue for local Ki67 testing with 89% using sections with thickness of 2-5 microns. For the Ki67 test, the majority (65%) reported using an in vitro diagnostic assay, 23% used a validated test, and 8% utilized a research-use-only assay. Ki67 antibody selection varied among the labs with 46% using the MIB-1 mouse monoclonal (Dako Agilent), followed by 33% using the 30-9 rabbit monoclonal (Ventana) and 12% reporting the SP6 rabbit monoclonal (Thermo Fisher). A majority (65%) reported using single pathologist visual assessment for scoring, and 17% reported using two or more pathologists. Use of automated digital image analysis (ADIA) was reported by 18% of labs, either alone or in combination with pathologist visual assessment. A significant portion (75%) reported using the International Ki67 in Breast Cancer Working Group (IKWG) recommendations, whereas 7% reported using only digital image analysis (e.g. Ventana Virtuoso). A minority (7%) indicated neither and instead described variations of “eyeball” or “hot spot” visual estimates. Most labs (65%) reported counting at least 500 cells with 15% of these counting more than 1000 cells. Remaining labs (30%) counted less than 500 or no cells. Predominantly, 85% reported counting cells in at least 3 or more high power fields. Most labs (96%) report Ki67 scores as a percentage of positive nuclei and the remaining minority reported using other methods (e.g. ranges [&lt; 10%, 10-20%, etc.] or H-score [0-300]). CONCLUSIONS The survey results suggest high global variability of local Ki67 testing practices with the highest variability observed in the test validation status, Ki67 clone, and scoring methods. Despite efforts by the IKWG to harmonize and increase the clinical validity of Ki67 as a biomarker, many labs indicating IKWG compliance had survey answers that were discordant with the specific guidelines set forth by the working group. Taking into account the totality of all answers provided by each respondent, only 51% of the surveyed labs fully conformed to the IKWG recommendations. Moreover, a small fraction conducts global estimations without specific cell counting or use “hot spot” scoring methods, despite the high variability and low reproducibility of these scores both intra- and inter-lab. This study demonstrates the benefits of using a central assay in clinical studies to reduce the variability of local Ki67 results in identifying high risk HR+ BC patients and suggests more work is needed to streamline the analytical practices of local Ki67 methodologies, which may directly impact clinical decisions such as the use of neoadjuvant therapies in HR+ BC.
Citation Format: Heather M. Moore, Wendy W. Lin, Tharu M. Fernando, Celine Lopez, Emma Kent, Karine Ellouk, Jennifer M. Giltnane. Variations in diagnostic practice of Ki67 scoring suggest standard guidelines needed for pathological assessment: survey results of global Ki67 testing methods [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-04-09.