Activation of the PI3K/AKT and MAPK/ERK pathways by various molecular mechanisms is ubiquitous in a variety of malignancies, including high grade astrocytomas [18
]. Our study evaluated the association of alterations of these pathways with various PA subsets. In recent years, several studies have highlighted the importance of MAPK/ERK activation in the pathobiology of PA. Activation occurs most frequently by alterations in the BRAF
gene, particularly a tandem duplication of its kinase domain resulting in a BRAF:KIAA1549
gene fusion [15
]. More recently, a similar duplication–fusion event was reported in a minority of PA involving RAF1
]. Other less specific alterations reported in PA include the BRAFV600E
point mutation, which leads to constitutive activation of BRAF. BRAFV600E
may more often be found in pediatric diffuse gliomas [27
] and gangliogliomas [6
]. The BRAFV600E
mutation also occurs in melanoma and papillary thyroid carcinoma.
In PA developing in the context of NF1 disease, homozygous inactivation of the NF1
gene is an alternative mechanism for MAPK/ERK activation and seems to be a mutually exclusive alternative to BRAF
]. Although NF1-associated PA seems to be associated with a better outcome when compared with its sporadic counterpart, associations of BRAF
aberrations with prognosis have been less consistent [8
]. Of note, 28% of the histologically anaplastic PA in our series were NF1-associated and not related to the optic pathways. Although NF1-associated PA tend to have a better outcome when compared with their sporadic counterparts, this is highlighted more in optic pathway tumors, which may even spontaneously regress, and are usually not resected in most instances at the current time [20
]. The optic pathway, at the biologic level, represents a unique niche in NF1 [33
], with a low potential for clinical and histological progression, explaining in part the absence of these tumors in our series. However, infiltrating and high grade astrocytomas may occur throughout the neuraxis in NF1, especially in older patients, and be associated with similar molecular abnormalities and adverse outcome as their sporadic counterparts [11
]. Another possible explanation for the increased prevalence of NF1 cases in our series is that they are predominantly consultation derived, and NF1-associated tumors other than PA, especially with unusual morphologies, are more likely to be shared in consultation.
It is of interest that BRAF
duplications were identified in 63% of the cerebellar anaplastic PA, but in none of seven non-cerebellar examples. This may suggest that non-cerebellar anaplastic PA may activate the ERK pathway by alternative mechanisms, in spite of similar histology, which may involve, among other alterations, point mutations (e.g. RAS
) or RAF1
rearrangements as previously described in a minority of PA [16
]. In addition, only 40% of the sporadic non-anaplastic PA in our series showed BRAF
duplication, although this alteration was present in almost half (47%) of posterior fossa examples. Variation in BRAF
aberration frequency according to anatomic site has been reported, with prior studies showing a decreased frequency of BRAF
rearrangements in non-cerebellar examples [34
], down to 38% in one study [12
]. In addition, BRAF
rearrangements may also be associated with lower patient age [12
], which can in part explain the lower prevalence in our relatively older patient population.
Although clinical observations and prior studies both noted aggressive clinical behavior in a subset of PA showing otherwise conventional histology [5
], molecular abnormalities associated with the worse outcome remained elusive, comprehensive gene expression studies demonstrating no global changes reliably distinguishing conventional from clinically aggressive PA [28
]. However, alterations in specific genes and proteins associated with a clinically aggressive phenotype were noted including increased levels of matrilin-2 [29
] and decreased levels of ALDH1L1 [23
Recent studies have also explored the relationship of various molecular alterations and outcome in PA. Tibbetts et al. [32
] did not find an association with recurrence free survival in PA with respect to ERK or mTOR activation. However, their study did not include histologically anaplastic PA, which had the highest degree of pS6 staining in our study, and AKT activation (pAKT) was not tested. At difference with our study, Horbinski et al. [12
] found homozygous p16
deletions by FISH in 6% of PA and LOH at the PTEN
locus in 50% by PCR-based microsatellite LOH analysis, findings not associated with adverse outcome. Their study also did not include histologically anaplastic PA, and the prevalence of homozygous p16
deletions was higher in this cohort (20%) in our study. With respect to PTEN
, we did not find heterozygous deletions by FISH in non-anaplastic PA, but in 32% of histologically anaplastic PA. These findings suggest that even though PTEN
LOH may be found in conventional PA, large deletions identified by FISH may be more closely associated with anaplastic histology.
Amplifications of receptor tyrosine kinases are important genetic events in the development of high-grade astrocytomas. EGFR
amplification is most frequently seen in primary glioblastomas of adults, while PDGFRA
amplification has been identified in a subset of pediatric high-grade astrocytomas [21
]. In our study, we did not encounter these aberrations.
In the current study, we demonstrate that, in addition to MAPK/ERK pathway activation, increased PI3K/AKT activity also seems to be a feature of clinically aggressive PA and particularly histologically anaplastic PA. A possible model summarizing these findings is illustrated in . Notably, however, NF1-associated tumors, including PA, are associated with increased mTOR pathway signaling [4
]. In addition to aberrations of MAPK/ERK, histologically anaplastic PA develop alterations characteristic of high grade astrocytomas, including heterozygous PTEN/10q
and homozygous p16
deletions. Frequent alterations in the PTEN/PI3K/AKT pathway were also noted in a prior study of malignant transformation in pediatric low grade gliomas [3
], although only one example progressed from a PA.
Fig. 7 Model of molecular mechanisms underlying PA and histologically anaplastic PA. RAS/BRAF/MEK pathway activation by various mechanisms is a key feature of sporadic PA and NF1 PA. Histologically anaplastic PA develop in addition hyperactivation of the PI3K/PTEN/AKT (more ...)
Although the status of the remaining PTEN
allele was not tested in our study, decreased expression of the gene at the mRNA level in histologically anaplastic PA suggests that this aberration may have biological significance. Furthermore, it seems to occur early in their evolution, being present in two (of 2) cases in which the PA precursors were tested. Our findings also reinforce recent findings highlighting the importance of PTEN
loss in malignant progression of other NF1-associated tumors, particularly neurofibromas progressing to malignant peripheral nerve sheath tumors [10
]. Now, in the current study, we were unable to demonstrate a definite relationship between PTEN
gene underexpression and pAKT/pS6 immunohistochemistry, even though each of these variables was more prevalent in histologically anaplastic PA. This suggests that alternative mechanisms to PTEN
loss may also activate AKT signaling in histologically anaplastic PA, in a similar way to other astrocytomas that have mutations in other PI3K pathway components [18
]. Future studies should be more illustrative in this regard.
PI3K/AKT activation underlies increased cellular proliferation characteristic of anaplasia and an adverse histologic phenotype in PA. Indeed, an inverse relation between MIB1-labeling index and progression-free survival was noted in one prior study of PA [2
]. Interestingly, a worsening of prognosis with increased MIB1 labeling was not confirmed by other studies [5
]. We did not identify a significant difference in MIB1-labeling indices between clinically aggressive/recurrent PA and conventional PA, but did find a significant increase in histologically anaplastic PA. These findings support the notion that increased cellular proliferation is an important, defining trait of histologic anaplasia in PA and a factor underlying the worse prognosis associated with these tumors.
In summary, this study assessed the prevalence of key molecular genetic abnormalities in clinically and histo-logically aggressive PA, and highlighted important differences in PA subsets with respect to PI3K/AKT activation status. Further, more comprehensive studies should confirm these findings and identify additional molecular aberrations in aggressive PA subsets, perhaps identifying therapeutic targets beneficial to astrocytoma patients in general.