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)
	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(

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.

Phosphohistone H3 expression is a strong prognostic indicator in node-negative breast carcinoma patients less than 55 years old treated with adjuvant chemotherapy4:

PHH3 expression per 10 HPF	10 year survival	20 year survival
<13	96%	96%
>=13	80%	58%

Diagnostic utility

Counting mitoses, including:

Grading of meningiomas1
Counting of mitotic figures in breast carcinoma2 and prediction of prognosis4.

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

4 Skaland I, Janssen EA, Gudlaugsson E, et al. Phosphohistone H3 expression has much stronger prognostic value than classical prognosticators in invasive lymph node-negative breast cancer patients less than 55 years of age. Mod Pathol 2007; 20:1307-15

This page last revised 6.4.2008.