Although previous studies have dealt with PPH3 staining of colorectal adenocarcinomas and ovarian serous adenocarcinomas,18,19
we are aware of only one study that focused on a tumour type, meningiomas, which requires a histoprognostic grading.15
This is why we focused on a tumour type in which the mitotic count is relevant to the prognosis—that is, breast adenocarcinomas. Our study showed a very good correlation between the mitotic figure count assessed with PPH3 labelling and that assessed by standard H&E sections. PPH3 mitotic figure count has several advantages. Firstly, it allows clear and unambiguous identification of mitotic figures, especially of prophase nuclei, a finding supported by the nuclear coexpression of PPH3 and cyclin B1, and consistent with previous results.19
As a consequence, PPH3 labelling improves the mitotic count. Accordingly, the histoprognostic grade of breast adenocarcinomas was shifted from grade I to grade II in one case, and from grade II to grade III in two cases. In addition, PPH3 staining is also a useful marker in tumours in which a high degree of apoptosis and necrosis can be misinterpreted in H&E staining. Furthermore, it allows a rapid detection of the area of highest mitotic activity, even at low‐power view. Interestingly, it may be useful to detect and count mitotic figures more precisely on small biopsy specimens that are sometimes crushed or contain a dense distorted tumour infiltrate.
We also observed some non‐mitotic (interphasic) cells labelled with anti‐PPH3 antibody, which displayed a finely granular staining. Juan et al12,13
found the same weak and punctate expression of PPH3 in interphasic leukaemic cells. Wolffe and Hayes20
also described interphase cells with a low level of histone H3 phosphorylation, which was interpreted as chromatin modification related to gene transcription. On the basis of cyclin‐B1 staining, these non‐mitotic (interphasic) cells could be divided into two classes: cells in the late G2 phase of the cell cycle (cytoplasmic cyclin‐B1 positive) and cells out of the G2 phase (cyclin‐B1 negative). Whatever the explanation of this immunolabelling of interphase nuclei, the important point is that these nuclei can be easily distinguished because of their specific staining pattern. In this context, further work is needed to explore the biological significance of this histone H3 phosphorylation out of the M phase.
- PPH3 staining allows clear identification of mitotic figures, especially prophase nuclei.
- In some tumours, PPH3 staining is useful to distinguish mitotic figures from morphological mimics, for example, nuclei of cells undergoing apoptosis or necrosis.
- PPH3 staining allows a rapid detection of the area of highest mitotic activity, even at low‐power magnification.
- This study demonstrates the feasibility of a computer-assisted determination of the mitotic index using PPH3 staining and image‐analysis software.
Only a few reports described an automatic counting of immunostaining signals. For example, counting of fluorescence in situ hybridisation signals in tissue sections showed a strong correlation between visual and automatic counting after removal of artefacts and noise with image‐processing techniques.21
In the present study, the good correlation between visual and computer‐assisted counts of PPH3‐positive mitotic figures with confocal microscopy suggests that this new automatic method is rapid, objective, reproducible and less time consuming for determining the mitotic index in tumours. This method should be promising in the future, and may be useful in grading other tumour types in which the mitotic index has also been shown to have prognostic significance. For a more precise automatic mitotic index determination, however, a specific method for differentiating nuclei of tumour cells from those of stromal cells needs to be developed.