NF1 associated low grade astrocytomas are predominantly pilocytic in type. Prior studies have indicated that a subset of low grade astrocytomas are difficult to classify, but offer no explanation of phenotypic variations in terms of possible underlying molecular mechanisms. As would be expected, the most conspicuous ultrastructural finding within all tumors in this study was the presence of numerous intermediate filaments, typical of astrocytic neoplasms. In our study, the majority of tumors available for ultrastructural review were PA. Most demonstrated typical features one would expect, such as Rosenthal fibers and intermediate filaments. In the GG intermediate filaments were present only within phenotypically glial cells. As expected, dense core granules were abundantly present in the GG, but were also sparsely represented in all three LGSI examined. Rosenthal fibers not appreciated on light microscopy were present ultrastructurally in LGSI as well as the single diffuse astrocytoma, thus suggesting morphologic similarities, at least at the ultrastructural level, among all low grade astrocy-tomas in NF1. Instead, the presence of ultrastructural features usually associated with a neuronal phenotype, including aligned micro-tubules and dense core granules, was typical, albeit an unexpected feature, of LGSI. These findings within low grade astrocytomas may represent phenotypic divergent differentiation in the setting of NF1.
A partial neuronal phenotype was also demonstrated by gene expression analysis (in a single tumor) and by increased expression of neuronal markers at the protein level. However, the predominant line of differentiation of these tumors remains glial, as reflected by strong consistent GFAP immunoreactivity and intermediate filament accumulation in all tumors. Classic neuronal and glioneuronal tumors have also been described in the setting of NF1, including gan-gliogliomas [1
], DNET like lesions [16
] and even desmoplastic infantile gangliogliomas [1
]. Unlike these lesions, our LGSI subset exhibits a predominant glial phenotype with only partial divergent neuronal differentiation, as described in other astrocytomas, including subependymal giant cell astrocytoma and pleomorphic xan-thoastrocytoma [17
The morphology of this low grade astrocytoma subset is somewhat reminiscent of another syndrome-associated tumor, i.e. subependymal giant cell astrocytoma. The features include nucleolar prominence, abundance of cytoplasm, expression of neuronal markers, and favorable prognosis [18
]. Subependymal giant cell astrocytoma is nearly restricted to patients with tuberous sclerosis, a syndrome arising as a consequence of TSC1
germline mutations. The molecular consequence is activation of the mTOR pathway [10
]. mTOR activation may also be an important biological event in epilepsy associated glioneuronal tumors [19
Increased mTOR activation has been shown to occur in NF1 deficient astrocytes and in 4 of 6 NF1-associated PA in a prior study [8
]. Curiously, in our group of NF1-associated low grade astrocytomas mTOR activation, as reflected by phospho-S6 and phospho-p70S6K levels, was frequent, but seemed to be higher in the low grade astrocytomas with peculiar cytologic features. Phospho-S6 levels are increased as a result of activation of mTOR which leads to phosphorylation of p70S6 kinase and thus activation of ribosomal S6, which leads to increased protein synthesis. The result may be increased cell size. These findings support the notion that some low grade astrocytomas represent a distinct morphologic subset of NF1-associated tumors. Although no obvious clinical differences are noted with respect to NF1-associated PA, our findings could be clinically relevant since the mTOR pathway may be pharmacologically targeted for therapeutic benefit [20
MAPK/ERK activation, as demonstrated by phospho-ERK immunoreactivity, was uniform in all tumors studied. This is expected, given that NF1 loss results in persistent activated RAS and downstream signaling through the MAPK/ERK pathway[8
rearrangements and mutations represent an alternative mechanism of MAPK/ERK activation in sporadic PA [21
]. It is of interest that prior studies have found BRAF
alterations to be absent in NF1-associated astrocytomas [22
], including 7 PA and 1 LGSI used in the current study. Therefore, BRAF
testing is not useful in distinguishing these subtypes of low grade astrocytoma in the setting of NF1.
One caveat is that not all LGSI demonstrate this unique morphology, and some may in fact represent other, inadequately sampled histologies, such as PA [1
]. Indeed, the possibility of under-sampling resulting in an erroneous diagnosis is a consideration whenever tumors with overlapping morphologic and ultrastructural features are encountered.
Our study suggests that NF1-associated PA and LGSI share some phenotypic features at the ultrastructural level, particularly Rosenthal fiber formation. However, the presence of occasional dense core granules and microtubules in addition to increased expression of neuronal associated genes, may represent divergent differentiation of some low grade astrocytomas in comparison with classic PA, the most prototypic NF1 associated astrocytoma. Furthermore, increased mTOR activation may be responsible for larger cell size despite similar proliferative indices and clinical behavior. Our findings, perhaps underlying the unconventional morphology of these tumors, require further studies to shed light upon the biology and molecular mechanisms underlying NF1-associated glial tumorigenesis.