Although many children with PAs have indolent tumors that are essentially cured by gross total surgical excision, a significant number of patients have tumors that are not amenable to complete resection but recur or disseminate. To date, few definitive histopathologic, biologic, or molecular markers have been identified to stratify the patients who may need more aggressive therapy or follow-up. Recent publications have evaluated the potential predictive value of histologic features of pediatric PAs but with discrepant, inconclusive results. For example, necrosis, vascular hyalinization, and calcification have all been suggested to predict an adverse outcome in one study and not in another.19,21
Only the presence of oligodendroglial-like morphology was consistently predictive of poorer outcome in these 2 studies. Another publication found that increased microvessel density, as determined by quantified immunohistochemistry, was associated with a significantly higher rate of progression in 41 pediatric patients with optic pathway gliomas.22
This interesting observation warrants further evaluation in PAs in other locations.
Tumor location has been well established as an adverse predictor of PFS previously and our study again confirmed this with a significantly higher proportion of tumors in the optic/hypothalamic area showing progression.
This study reconfirmed the validity of using the MIB-LI as an important independent predictor of PFS in patients with incompletely resected PAs. None of the other immunohistochemical markers studied here was of use in predicting PFS in this large and unique series of patients with incompletely resected tumors and adequate clinical follow-up.
The use of MIB-1 immunohistochemistry has become well established in diagnostic neuro-oncologic pathology to idntify tumors with a large population of proliferating cells, which usually indicates more aggressive biologic behavior and a poorer patient outcome. The MIB-1 monoclonal antibody is one of the most frequently used and sensitive of the commercially available Ki67 antibodies, with over 4000 literature citations. Although the function of the Ki67 antigen is still unknown, this nonhistone nuclear protein is expressed throughout the active parts of the cell cycle (G1, S, G2, and mitosis).23
The most frequent criticism of the use of MIB-1 immunostaining is the variable intraobserver reproducibility depending on the method used for quantification.24
Our method of quantification is the one most widely used and is the same as was performed in our previous study.2
As the same counting methodology was used for the analysis of all 3 of the proliferative markers in this study, this provides a useful comparison of their benefits.
PHH3 is becoming well established as a prognostic marker for identifying and stratifying some types of adult brain tumors, as it allows easy identification of mitosis. While counting mitoses has long been a method of identifying proliferative activity in neoplastic cells, highlighting the mitotic structures with immunoreactive labels such as PHH3, makes the practice quicker and more accurate by eliminating technical artifacts and allowing the discrimination of mitoses from apoptotic cells. Our study indicates, however, that in PAs, where mitoses are very uncommon, quantification of PHH3 is not useful in identifying patients with shortened PFS. More than half of our cases had no PHH3-positive mitoses identified in the histology sections. In cases that had PHH3-immunoreactive mitoses, the number of reactive cells did not discriminate between patients who had stable disease or clinical progression. This suggests PHH3 immunohistochemstry may be of more benefit in high-grade malignancies, where mitoses are more numerous.
MCM2 is another proliferative marker that holds promise as a potential prognostic variable in cancer histopathology. MCM2 is one of a family of proteins that form a complex that appears to function as a DNA helicase, allowing DNA to unwind at the initiation of DNA replication.11
In normal nonneoplastic tissues, immunohistochemical expression of MCM2 is restricted to the nuclei of cells in proliferating areas, such as the basal regions of gastrointestinal and squamous epithelia.8
In carcinomas, a higher proportion of poorly-differentiated tumor cells express MCM2 than do well-differentiated tumor cells.8
Our study found tumors had a smaller proportion of positive cells with MCM2 than were seen with the MIB-1 staining; however, the 2 values were statistically related (P
= .002). Due to the diminished degree of reactivity, however, MCM2 staining was not an independent predictor of PFS. It could be anticipated that MCM2 would stain a smaller proportion of tumor cells than MIB-1, as it is known to selectively identify cells in the early initiation phase of DNA replication and is downregulated in differentiated or quiescent cells.10,11
It is interesting that several studies have shown MCM2 staining to be more abundant than MIB-1 in a variety of normal and neoplastic tissues.8,10,11
In meningiomas in adults, MCM2-positive cells were often more than 4 times as prevalent as MIB-1 reactive cells, although it was reported that there was marked heterogeneity of MCM2 staining in different areas of the tumor.10
That study reported the majority of recurrent meningiomas had an MCM2-LI of >30%, a number far exceeding the degree of staining seen in any of our gliomas.10
A study examining MCM2 expression in a series of oligodendrogliomas also found a 4 times higher proportion of tumor cells staining for MCM2 than MIB-1, with a mean of 20% of the tumor cells showing reactivity for MCM2.11
It is not clear why in both those studies such a high percentage of tumor cells expressed this supposedly tightly regulated marker of DNA replication. It would be anticipated, as was seen in our study, that a smaller number of cells expressed MCM2 than MIB-1. It is possible that reactivity for MCM2 varies among different types of CNS tumors, and further studies of glial neoplasms with this marker may be of benefit.
Identifying specific biomarkers that are predictive of therapeutic response holds the potential to further improve therapy specifically tailored to the individual patient. A growing library of immunohistochemical reagents is now available to help guide therapeutic decisions. Immunohistochemical assays have the advantage over other types of tests in that they are readily available in most hospital laboratories and the studies can be done on routinely processed tissue sections in conjunction with the diagnostic pathology workup. One such potential biomarker, MGMT, has been studied fairly extensively in adult malignant gliomas, as it holds the potential to predict tumor sensitivity to alkylating agent chemotherapy.13,14
In children with high-grade gliomas, MGMT expression by immunohistochemistry strongly correlated with outcome in patients treated with alkylator-based adjuvant chemotherapy.15
As there have been suggestions that MGMT expression may be an independent predictor of outcome, regardless of the use of adjuvant therapy, we thought it was important to evaluate it in PAs. For our study, we selected a previously published, detailed scoring system to semiquantitatively estimate the amount of MGMT immunoreactivity, as this protein is known to be heterogeneous in its distribution and intensity in tumors.13
In addition to quantifying random fields, as had been previously described, we also examined a series of contiguous fields from the so-called hot-spot areas of greatest intensity to see if either method was predictive of PFS. Although there is great variability in the degree of MGMT expression among different PA cases, the amount of reactivity did not correlate with PFS. To the best of our knowledge, only one prior study has examined MGMT by immunohistochemistry in pediatric PA cases and, although it used a much simpler grading scheme than we did, it also found no association of MGMT expression with PFS.15
The lack of association of MGMT expression and PFS provides additional evidence that PAs are biologically distinct from high-grade infiltrating gliomas, and a search for other relevant biomarkers should continue.
Relatively little is understood about the molecular changes that prompt the development of PAs, and it is expected that better understanding of these alterations will identify prognostically relevant markers and potential therapeutic targets. High-sensitivity comparative genomic hybridization analysis has found a few common alterations, such as gains in chromosome 7q34 leading to increased copy number of the BRAF
gene, in the majority of cases.25
The clinical and prognostic significance of these findings is not yet determined.
In conclusion, this study reconfirms the value of the MIB-1 labeling index as a predictor of outcome in patients with PAs and indicates that more selective markers of cell proliferation, such as PHH3 and MCM2, are not useful in determining progression in this neoplasm. The amount of MGMT expression does not show promise as a predictor of PFS. Until further details of the molecular pathways involving PA are delineated, the MIB-1 labeling index shows the highest promise in predicting which patients are at highest risk of disease progression.