Phosphohistone-H3, PHH3

Histone H3 is a core histone protein forming a major constituent of chromatin. Serine-10 and serine-28 are only phosphorylated during mitosis. An antibody has been raised that only recognises the phosphorylated form of histone H3 (PHH3). Since the histone is not phosphorylated during apoptosis, anti-PHH3 allows genuine mitoses to be distinguished from apoptotic nuclei. Pyknotic and distorted nuclei are negative. Small round early prophase nuclei are positive, but easily distinguished from other M-phase nuclei and are excluded from the mitotic index. There is a suggestion that immunoreactivity declines with prolonged storage¹. Staining may be unsatisfactory in large specimens, such as breast lumpectomies and mastectomies, due to delayed fixation³.

Immunohistochemical expression

PHH3 stains mitotic figures from early prophase through metaphase, anaphase and telophase³. Staining is seen on the condensed chromosomes³. Scattered interphase nuclei may show speckled positivity³. There is non-specific staining of the cytoplasm of macrophages, neutrophils and mast cells³.

Both PHH3 and MPM-2 result in higher mitotic counts than are obtained with H/E sections. In one study across a range of tumours, the average mitotic counts per 10 HPF were 5.9 ± 15.8, 10.1 ± 22.6 and 11.0 ± 27.3 for H/E, PHH3 and MPM-2 staining respectively³.

PHH3 differentiated benign, atypical and anaplastic meningiomas¹:

number of cases

mitoses/10HPF mean (range)

assessed on H/E

assessed using PHH3

Benign

26

1.4 (0-3)

2.2 (0-7)

Atypical

20

9 (4-17)

16 (2-55)

Anaplastic

8

22 (20-35)

34 (10-93(

Using the WHO criteria established for mitotic indices on H/E sections would result in the upgrading of a significant number of meningiomas: the mitotic count needs to be recalibrated for the more sensitive method.

Diagnostic utility

Counting mitoses, including:

Grading of meningiomas¹
Counting of mitotic figures in breast carcinoma²

References

¹ Ribalta, T., I. E. McCutcheon, et al. (2004). "The mitosis-specific antibody anti-phosphohistone-H3 (PHH3) facilitates rapid reliable grading of meningiomas according to WHO 2000 criteria." Am J Surg Pathol 28(11): 1532-6.

² Baehner R, Weidner N. Enhanced mitotic figure counting in breast carcinomas using mitosis-specific antibody: anti-phosphohistone-H3 (PHH3). Mod Pathol. 2000;13:17A.

³ Tapia C, Kutzner H, Mentzel T, et al. Two Mitosis-Specific Antibodies, MPM-2 and Phospho-Histone H3 (Ser28), Allow Rapid and Precise Determination of Mitotic Activity. Am J Surg Pathol 2006; 30:83-89

This page last revised 21.1.2006.

	number of cases	mitoses/10HPF mean (range)
	number of cases	assessed on H/E	assessed using PHH3
Benign	26	1.4 (0-3)	2.2 (0-7)
Atypical	20	9 (4-17)	16 (2-55)
Anaplastic	8	22 (20-35)	34 (10-93(