Abstract P5-02-11: Assessment of tumor proliferation by mitotic activity index, phosphohistone H3 and Ki67 in early stage ER+/Her2- breast cancer
Authors: van Steenhoven Julia E.C. , Kuijer Anne , van Leeuwen A. M. , van Gorp Joost M. , van Diest Paul J. , van Dalen Thijs 
Published: March 2020

Abstract
Introduction: Determining expression of tumor proliferation by mitotic index or Ki67 expression is one of the strongest prognostic factors in breast cancer, but its reproducibility is not optimal. Recently, phosphohistone H3 (PHH3) was proposed as a novel marker specific for mitosis. For the present study, we hypothesized that counting PHH3 positive cells would be more reproducible than mitoses counting in H&E sections (MAI) and Ki67.
Methods: Tumor samples of 159 early breast cancer patients were immunohistochemically stained for Ki67 and PHH3. MAI and PHH3 scores were assessed by three breast cancer dedicated pathologists. Ki67 scores were assessed separately by two breast cancer pathologists. We used for Ki67 a threshold of 20% and for PHH3 the threshold score was 13. Intraclass correlation coefficient (ICC) and Cohen’s κ statistics were used to assess inter-observer agreement. The impact of swapping mitotic index with PHH3 in histologic grading was assessed.
Results: The ICCs of PHH3 as a measure for inter-observer agreement on a continuous scale were 0.78 (obs. 1 vs. 2), 0.76 (obs. 2 vs. 3) and 0.97 (obs. 1 vs. 3). The PHH3 kappa scores (κ 0.78, 0.80, 0.68, table 1) were contrasted by fair to moderate inter-observer variation for Ki67 and MAI: Ki67, k 0.55 (obs. 1 and 2) and MAI k 0.38 (obs. 1 vs. 2), k 0.26 (obs. 2 vs. 3), k 0.56 (obs. 1 vs. 3). When using PHH3 in the Nottingham grading score instead of MAI, variation in histologic grading decreased (MAI k 0.43, 0.35, 0.32 vs. PHH3 k 0.52, 0.48, 0.52), while the proportion of grade III tumors increased (table 2).
Conclusion: PHH3 seems to outperform Ki67 and MAI as a reproducible measure for tumor proliferation in breast cancer. Variation in histologic grading might be improved by using PHH3, but the resulting increase of high-grade cancers remains to be addressed.
Table 1 The inter-observer agreement of PHH3 scored by three different breast cancer pathologistsObs. 2Obs. 3Obs. 3Obs. 1PHH3&lt;13PHH3 ≥13PHH3&lt;13PHH3 ≥13Obs. 2PHH3&lt;13PHH3 ≥13PHH3&lt;13583905PHH3&lt;13597PHH3 ≥138371054PHH3 ≥13931Kappa0.780.800.67Abbreviations: PHH3, phosphohistone H3, Obs, observer.The concordance between obs. 1 & 2, obs. 1 & 3 and obs. 2 & 3 was 90%, 91% and 85%, respectively.
Table 2. The impact of swapping mitotic index with PHH3 in histologic gradingGrade I n(%)Grade II n(%)Grade III n(%)Observer 1H&E42(26)104(65)13(8)PHH330(19)77(48)52(33)Observer 2H&E35(33)62(58)9(8)PHH323(22)57(54)26(24)Observer 3H&E25(16)101(64)33(21)PHH320(13)90(56)49(31)Abbreviations: PHH3, phosphohistone H3, H&E, hematolylin and eosin
Citation Format: Julia E.C. van Steenhoven, Anne Kuijer, A. M. van Leeuwen, Joost M. van Gorp, Paul J. van Diest, Thijs van Dalen. Assessment of tumor proliferation by mitotic activity index, phosphohistone H3 and Ki67 in early stage ER+/Her2- breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-02-11.