Objectives Anti-phosphorylated histone H3 (pHH3) antibodies specifically detect the core protein histone H3 only when phosphorylated at serine 10 (Ser10) or serine 28 (Ser28). Measurement of pHH3 levels can be used for quantifying mitosis and the effectiveness of mitotic inhibitors in early drug development. However, data on the expression level of pHH3 (Ser10) and pHH3 (Ser28) among different cancers are limited. This study was designed to investigate the expression levels of pHH3 across different types of cancers, using uniform techniques and assay platforms in a single laboratory.

Design Retrospective study.

Setting Single laboratory.

Specimens Formalin-fixed, paraffin-embedded various human cancer specimens were provided by Mosaic Laboratories Tissue Bank.

Primary and secondary outcome measures Using immunohistochemistry, pHH3 levels were measured using both pHH3 (Ser10) and (Ser28) antibodies among 10 human melanoma and 10 ovarian tumour samples. The samples were reviewed blindly by two reviewers. pHH3 (Ser10) was then selected to measure the pHH3 levels in cancers of breast, colorectal, oesophageal, gastric, head and neck and lung (n=5 for each cancer).

Results The pHH3 (Ser10) expression was higher than pHH3 (Ser28) in both melanoma and ovarian cancers (p<0.01), with the mean (SD) levels of 1.28% (0.47%) for Ser10 and 0.53% (0.44%) for Ser28 among melanoma and 3.47% (3.51%) for Ser10 and 0.62% (0.68%) for Ser28 among ovarian cancers, respectively. No statistically significant differences were observed among different cancer types tested for pHH3 using Ser10 (p=0.197). No reviewer effect was identified.

Conclusions The pHH3 Ser10 was significantly higher than Ser28 and may serve as the more robust of two pHH3 assays for measuring mitotic index.