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Pilocytic astrocytoma (PA) is the most frequent primary brain tumor developing in patients with neurofibromatosis type 1 (NF1) syndrome. It is also known that the whole spectrum of glial neoplasia may develop in these patients, including low-grade astrocytomas, which are notably difficult to classify, and overtly diffuse gliomas, which are histologically indistinguishable from those occurring sporadically (e.g., diffuse astrocytoma, anaplastic astrocytoma, and glioblastoma) [6]. However, the specific genetic drivers of these tumors are not completely characterized in contrast to gliomas developing in a sporadic setting [1]. Prior studies have linked alterations in the alpha thalassemia/mental retardation syndrome X-linked (ATRX) or death domain-associated protein (DAXX) genes with the alternative lengthening of telomeres (ALT) phenotype in a subset of cancers [3]. ATRX alterations predominate, but ATRX and DAXX are chromatin-remodeling proteins that cooperate at repetitive regions (e.g., telomeres) to incorporate H3.3 into heterochromatin. ATRX mutations and ALT are also associated with specific molecular subgroups of brain tumors, particularly a subset of pediatric glioblastoma [7] and IDH mutant diffuse astrocytomas [1]. Here, we hypothesized that ATRX alterations and concomitant acquisition of ALT may also play a role in NF1-associated gliomas. We tested for ALT using telomere-specific FISH, and nuclear ATRX/DAXX loss through immunohistochemistry in archival paraffin sections using previously published criteria [3]. Additionally, a subset of cases that were ambiguous by FISH due to high tissue autofluorescence were evaluated using a chromogenic (CISH) method (n = 9). A total of 27 gliomas from 26 patients with NF1 syndrome were successfully tested for these markers (ATRX n = 26, ALT FISH/CISH n = 26) (clinicopathologic and molecular data are outlined in supplementary Table 1). The diagnosis of NF1 was performed using established clinical criteria [5]. ALT was present in 10 of 26 (38 %) cases and ATRX loss in 9 of 26 (35 %) cases. DAXX expression was preserved in all 19 (100 %) cases tested. Interestingly, ALT was present in 8 (of 12) diffuse/high-grade gliomas compared to only 2 (of 14) low-grade circumscribed gliomas, while ATRX loss was identified in 7 (of 12) diffuse/high-grade gliomas compared to 2 (of 14) low-grade circumscribed gliomas, a statistically significant difference (p = 0.013 and p = 0.038, respectively, two-tailed Fisher’s exact test) (Fig. 1). IDH1 (R132H) immunohistochemistry was negative in 15 (of 15) cases (6/6 ALT+/ATRX−), p53 was positive in 10 (of 17) cases (6/8 ALT+/ATRX−) and H3 p.K27M was positive in 1 (of 9) case. There was high concordance between ALT and ATRX loss (22 of 25, 88 % cases). The two ALT-positive/ATRX-preserved discordant cases consisted of a low-grade astrocytoma with peculiar SEGA-like morphology (as previously described in NF1 patients) [6] and an anaplastic pleomorphic xanthoastrocytoma (A-PXA). For these cases, we next performed combined telomere-specific FISH for ALT and ATRX immunofluorescence. While we observed no ATRX loss in the low-grade astrocytoma, the A-PXA showed ATRX mislocalization, suggesting an altered protein, which consisted of distinct punctate foci, some of which co-localized with the ultrabright telomeric foci (Fig. 1d–f). When focusing on age, combined ALT+/ATRX− was present in only 1 of 13 pediatric cases (a glioblastoma) vs. 7 of 11 adult cases (p = 0.008). The frequency was particularly high in the adult diffuse/high-grade glioma group, with all but one gliosarcoma case (7/8) demonstrating the ALT phenotype and 6/8 of cases demonstrating combined ALT+/ATRX−.
In summary, our data support an important role for ATRX loss and acquisition of ALT in the biology of NF1-associated gliomas, particularly diffuse and high-grade tumors developing in adults. These findings expand the subset of glial neoplasms that demonstrate this unique genotype/phenotype correlation. It appears that ALT+/ATRX− facilitates gliomagenesis/glial progression in the context of putative early initiating genetic events, leading to specific glioma subgroups. In this study, the culprit is NF1 loss, while in other astrocytomas it is either IDH or histone (G34) mutations, with TP53 alterations being frequent in all of these subgroups. Future studies should elucidate the basic mechanisms for these alterations, and their potential as prognostic and predictive biomarkers, as well as therapeutic targets, as is being explored in other ATRX mutant gliomas [4] or ALT-positive cancers [2].
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The study was supported in part by Pilocytic/Pilomyxoid Fund, including Lauren’s First and Goal and the PLGA Foundation (FJR).
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Rodriguez, F.J., Vizcaino, M.A., Blakeley, J. et al. Frequent alternative lengthening of telomeres and ATRX loss in adult NF1-associated diffuse and high-grade astrocytomas. Acta Neuropathol 132, 761–763 (2016). https://doi.org/10.1007/s00401-016-1619-0
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DOI: https://doi.org/10.1007/s00401-016-1619-0