Improving mitotic cell counting accuracy and efficiency using phosphohistone‐H3 (PHH3) antibody counterstained with haematoxylin and eosin as part of breast cancer grading

BACKGROUND: Mitotic count in breast cancer is an important prognostic marker. Unfortunately, substantial inter‐ and intraobserver variation exists when pathologists manually count mitotic figures. To alleviate this problem, we developed a new technique incorporating both haematoxylin and eosin (H&E) and phosphorylated histone H3 (PHH3), a marker highly specific to mitotic figures, and compared it to visual scoring of mitotic figures using H&E only. METHODS: Two full‐face sections from 97 cases were cut, one stained with H&E only, and the other was stained with PHH3 and counterstained with H&E (PHH3–H&E). Counting mitoses using PHH3–H&E was compared to traditional mitoses scoring using H&E in terms of reproducibility, scoring time, and the ability to detect mitosis hotspots. We assessed the agreement between manual and image analysis‐assisted scoring of mitotic figures using H&E and PHH3–H&E‐stained cells. The diagnostic performance of PHH3 in detecting mitotic figures in terms of sensitivity and specificity was measured. Finally, PHH3 replaced the mitosis score in a multivariate analysis to assess its significance. RESULTS: Pathologists detected significantly higher mitotic figures using the PHH3–H&E (median ± SD, 20 ± 33) compared with H&E alone (median ± SD, 16 ± 25), P < 0.001. The concordance between pathologists in identifying mitotic figures was highest when using the dual PHH3–H&E technique; in addition, it highlighted mitotic figures at low power, allowing better agreement on choosing the hotspot area (k = 0.842) in comparison with standard H&E (k = 0.625). A better agreement between image analysis‐assisted software and the human eye was observed for PHH3‐stained mitotic figures. When the mitosis score was replaced with PHH3 in a Cox regression model with other grade components, PHH3 was an independent predictor of survival (hazard ratio [HR] 5.66, 95% confidence interval [CI] 1.92–16.69; P = 0.002), and even showed a more significant association with breast cancer‐specific survival (BCSS) than mitosis (HR 3.63, 95% CI 1.49–8.86; P = 0.005) and Ki67 (P = 0.27). CONCLUSION: Using PHH3–H&E‐stained slides can reliably be used in routine scoring of mitotic figures and integrating both techniques will compensate for each other's limitations and improve diagnostic accuracy, quality, and precision.