Yin Wang ¹ Zou Jiarui ² Qinghua Cao ¹ Guihong Dai ³ Panhong Fan ⁴ Xue Gong ⁵ Jinyan Jiang ⁶ Yanqing Kong ⁷ Chao Liu ⁸ Chunhui Liu ¹ Chenjia Lu ⁹ Meiren Li ¹⁰ Zhiqiang Lang ¹¹ Yang Lin ¹² Yan Peng ¹³ Haiyan Shi ¹⁴ Yuhuan Wang ¹⁵ Jiu Wang ¹² Bichen Xie ¹⁶ Bing Yang ¹ Guohua Yu ¹¹ Cuiping Zhang ¹ Hengming Zhang ¹⁷ Luting Zhou ¹⁸ Zilan Zhang ¹⁹ Zhenli Zhu ¹ Junmei Hao ^1*

• ¹ Yantai Affiliated Hospital of Binzhou Medical University, Yantai, Shandong Province, China
• ² Hangzhou Red Cross Hospital, Hangzhou, Zhejiang Province, China
• ³ Taizhou School of Clinical Medicine， Nanjing Medical University, The Affiliated Taizhou People's Hospital, Nanjing Medical University, Taizhou, China
• ⁴ Henan Provincial People's Hospital, Zhengzhou, Henan Province, China
• ⁵ Qinghai University Affiliated Hospital, Qinghai, China
• ⁶ Chenzhou No.1 people hospital, Chenzhou, China
• ⁷ Shenzhen Maternity and Child Healthcare Hospital, Shenzhen, Guangdong Province, China
• ⁸ (Guangdong Academy of Medical Sciences), Southern Medical University, Department of Pathology, Guangdong Provincial People's Hospital, Guangzhou, China
• ⁹ The people’s hospital of LongHua,ShenZhen, ShenZhen, China
• ¹⁰ Jiujiang University Affiliated Hospital, Jiujiang, Jiangxi Province, China
• ¹¹ Affiliated Yantai Yuhuangding Hospital, Qingdao University, Yantai, China
• ¹² School of Public Health ,Binzhou Medical University,Yantai,Shandong,China, Yantai, Shandong Province, China
• ¹³ The third affiliated hospital of soochow university/changzhou first people’s hospital, Changzhou, China
• ¹⁴ Guangdong Hospital of Integrated Traditional Chinese and Western Medicine, Foshan, China
• ¹⁵ People's Hospital of Xinjiang Uygur Autonomous Region, Ürümqi, Xinjiang Uyghur Region, China
• ¹⁶ Affiliated Hospital of Jiangnan University, Wuxi, Jiangsu Province, China
• ¹⁷ Weifang People's Hospital, Weifang, Shandong Province, China
• ¹⁸ Department of Pathology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, Shanghai, China
• ¹⁹ Department of Pathology,Northern Jiangsu People’s Hospital, Yangzhou, China

Purpose: Recommendations from National Health Commission of China (NHCC) and International Ki67 Working Group (IKWG) were issued respectively to guide immunohistochemistry (IHC)-based Ki67 scoring for breast cancer patients in daily clinical practice. They were evaluated in this multi-institutional study alongside with results from Quantitative Dot Blot (QDB) method. Method: Three alternative adjacent sections each from 40 primary ER+ breast cancer resection blocks were randomly assigned a number from 1 to 120 for Ki67 staining and reviewed by 21 pathologists while the other three alternative sections were sent for QDB analysis of Ki67 protein levels. Ki67 scores were grouped by 5/30% (IKWG), 10/30% (NHCC) and 20/30% (NHCC appendix 9, NHCCa9) respectively while QDB results were grouped by C5-C95 of 2.31 nmole/g defined in previous study as low, equivocal and high risk groups. Results: The overall Intraclass Correlation Coefficient (ICC) was 0.785 for IHC evaluations from 21 pathologists, with the Fleiss Kappa at 0.555, 0.628 and 0.480 when Ki67 scores were grouped by the guidance from IKWG, NHCC and NHCCa9 respectively. In comparison, the ICC and Fleiss kappa for QDB analysis were at 0.939 and 0.831. When IHC and QDB results were cross-referenced, more specimens were grouped as high risk by QDB than IHC, and NHCCa9 led to highest percentage of disagreement between two methods. Conclusion: The IKWG recommendation was harder to achieve categorized agreement among pathologists than that of NHCC, yet it led to best agreement with QDB to define low-risk group. QDB method offered significantly improved consistency over current IHC-based Ki67 assessment.