In astrocytomas proliferative activity has been positively correlated to tumour grade and prognosis. Small biopsies and intricate histology make diagnosing difficult, and reliable biological markers are highly needed. In this immunohistochemical study, we demonstrated positive correlation between the proliferation markers Ki-67/MIB-1, mitosin, survivin, pHH3, and DNA topoisomerase IIα. Furthermore, Ki67/MIB-1 and pHH3 indicated poorer survival in univariate analyses.
Mitotic activity is fundamental in the histopathologic grading of human astrocytomas. Identification of mitotic figures is hampered by several factors including squeezed cells in stereotactic biopsies, distortion and similarities to chromatin changes in apoptotic and pycnotic cells . We experienced such elements, particularly in small biopsies and in paraffin sections prior used in frozen sections diagnostics. In contrast to others [5, 6, 28, 29] we did not find positive correlations between mitoses and most of the proliferative markers, probably because of the above mentioned factors. Hence, this observation suggests that antibodies against proliferation-associated antigens are useful to obtain an optimal profile of the proliferative activity, especially in small brain tumour samples.
Ki-67/MIB-1 immunostaining worked well and yielded credible results in our study. The LIs displayed a wide range of values that overlap with indices in both grade II and grade IV astrocytomas , and is regarded as the main reason for Ki-67/MIB-1 not being included in the routine histopathological diagnosis of astrocytic tumours . Thus, this marker should not be used alone, but in combination with established histopathological criteria of malignancy.
Proposals for clinical threshold values have been suggested  without a consensus being reached. Using a value of 10% [3, 7] a significant impact on survival was found, verifying this as a reasonable threshold.
pHH3 is a novel promising proliferation marker in tumour pathology, however the experience in astrocytomas is limited. In our hands, this antibody provided reliable immunostaining with distinct nuclear positivity in cells with mitotic morphology. Positive correlations with the other proliferative markers were found except mitoses. This discrepancy may be due to the problem to identify mitotic figures in squeezed and small biopsies. Comparison between the number of mitoses and pHH3 positive nuclei, revealed the latter to be higher, in accordance with being a more sensitive marker for mitoses [5, 14, 29, 30]. A major drawback of the pHH3 immunostaining seems to be positivity in non-mitotic cells . This, together with the subjective determination of mitotic morphology, may lead to misinterpretation. We demonstrated that higher indices were associated with poorer survival in univariate analyses in accordance with others . For this reason pHH3 stands out as a reliable biomarker, however, larger studies are necessary to further elucidate this observation.
Survivin immunoreactivity was located both in the cytoplasm and in the nucleus, as described by others [6, 8, 18]. The nuclear positivity was in contrast to the cytoplasmatic staining, easily detectable and distinct. As the localization of survivin compartments in the nucleus is correlated to cell division and prognosis [6, 17, 19], only the nuclear positivity was included. Survivin expression showed significant positive correlation with all of the proliferation markers, suggesting that nuclear survivin positivity can be a reliable proliferation marker in astrocytic tumours. Our data demonstrated no correlation between survivin expression and survival. The literature is contradictory in this matter [8, 17–19], and larger studies are needed to clarify this topic. The effect of survivin on radiotherapy resistance is intriguing. If, however, radiotherapy effects depend on the presence or not of survivin, the implication in anaplastic astrocytomas can be limited, as all of the tumours in our material displayed positively stained cells.
The results from our TIIα immunostaining correlated well to the other proliferation markers, in agreement with previous reports [8, 11, 24]. The number of positively stained nuclei was easily calculated, and was in general of a lower value than the values of Ki-67/MIB-1, as seen in previous studies [11, 24]. This result could be explained by the different protein expression throughout the cell-cycle. TIIα positive cells in G0- and G1 phase have been reported , and may represent a drawback of this marker. Using the median of 4% as a cut-point, TIIα was significantly associated with survival in our material. This is in accordance with others using similar cut-points  and suggests that TIIα could be of prognostic value in astrocytic tumours.
Mitosin also displayed significant correlations to all of the proliferative markers, which is in accordance with studies on other malignancies . Immunoreactive nuclei were easily identified and can be explained by the mitosin expression in S-, G2-, and M phase, and its rapid degradation [25, 26]. Our study could not correlate mitosin expression significantly to survival.
Due to the small number of cases, the survival analyses have been carefully interpreted. However, univariate analyses have gathered sense of which markers that could be further investigated in well-designed studies for prognostic relevance.
The caveats of immunohistochemical analyses include different antibodies, different antigens, background staining, and inhomogeneous staining that can contribute to interlaboratory and interobserver variations. Wrong assumptions can also be made as the knowledge of the novel markers is limited. For instance, protein overexpression in tumour cells can represent genetic aneuploidity, mutated genes or increased transcription factors. Additionally, the functions of a proliferative marker may not exclusively be related to the cell cycle, as was the case for the proliferating cell nuclear antigen (PCNA) .
Variations in immunoreactivity may be due to different expression during the cell cycle. The recorded wide range of values represents a major drawback of such markers as one will experience considerable overlap between malignancy groups. Thus, one should consider the possibility to introduce a panel of proliferation markers to identify more aggressive astrocytomas.