Glioneuronal tumor with ATRX alteration, kinase fusion and anaplastic features (GTAKA): a molecularly distinct brain tumor type with recurrent NTRK gene fusions

Abstract

Glioneuronal tumors are a heterogenous group of CNS neoplasms that can be challenging to accurately diagnose. Molecular methods are highly useful in classifying these tumors-distinguishing precise classes from their histological mimics and identifying previously unrecognized types of tumors. Using an unsupervised visualization approach of DNA methylation data, we identified a novel group of tumors (n = 20) that formed a cluster separate from all established CNS tumor types. Molecular analyses revealed ATRX alterations (in 16/16 cases by DNA sequencing and/or immunohistochemistry) as well as potentially targetable gene fusions involving receptor tyrosine-kinases (RTK; mostly NTRK1-3) in all of these tumors (16/16; 100%). In addition, copy number profiling showed homozygous deletions of CDKN2A/B in 55% of cases. Histological and immunohistochemical investigations revealed glioneuronal tumors with isomorphic, round and often condensed nuclei, perinuclear clearing, high mitotic activity and microvascular proliferation. Tumors were mainly located supratentorially (84%) and occurred in patients with a median age of 19 years. Survival data were limited (n = 18) but point towards a more aggressive biology as compared to other glioneuronal tumors (median progression-free survival 12.5 months). Given their molecular characteristics in addition to anaplastic features, we suggest the term glioneuronal tumor with ATRX alteration, kinase fusion and anaplastic features (GTAKA) to describe these tumors. In summary, our findings highlight a novel type of glioneuronal tumor driven by different RTK fusions accompanied by recurrent alterations in ATRX and homozygous deletions of CDKN2A/B. Targeted approaches such as NTRK inhibition might represent a therapeutic option for patients suffering from these tumors.

Keywords: ATRX; DNA methylation; Gene fusion; Glioneuronal tumor; NTRK.

Fig.1

Unsupervised, non-linear t-distributed stochastic neighbor embedding (t-SNE) projection of DNA methylation array profiles from 738 tumor samples. DNA methylation profiling reveals a molecular distinct group of glioneuronal tumors (GTAKA). Reference DNA methylation classes: dysembryoplastic neuroepithelial tumor (DNET), rosette-forming glioneuronal tumor (RGNT), diffuse leptomeningeal glioneuronal tumor subtype 1 (DLGNT_1), diffuse leptomeningeal glioneuronal tumor subtype 2 (DLGNT_2), extraventricular neurocytoma (EVNCYT), papillary glioneuronal tumor (PGNT), ganglioglioma (GG), polymorphous low-grade neuroepithelial tumor of the young (PLNTY), myxoid glioneuronal tumor, PDGFRA-mutant (MYXGNT), diffuse glioneuronal tumor with oligodendroglioma-like features and nuclear clusters (DGONC), glioneuronal tumor kinase-fused (GNT_KinF_A), central neurocytoma (CN), cerebellar liponeurocytoma (LIPN), desmoplastic infantile ganglioglioma / desmoplastic infantile astrocytoma (DIG/DIA), angiocentric glioma MYB/MYBL1-altered (LGG_MYB), pilocytic astrocytoma hemispheric (PA_CORT), pilocytic astrocytoma infratentorial (PA_INF), pilocytic astrocytoma midline (PA_MID), pleomorphic xanthoastrocytoma (PXA), infant-type hemispheric glioma (IHG), high-grade astrocytoma with piloid features (HGAP), subependymal giant cell astrocytoma (SEGA), diffuse midline glioma, H3 K27-altered (DMG_K27), diffuse hemispheric glioma, H3 G34-mutant (DHG_G34), glioblastoma, IDH-wildtype, RTK1 subtype (HGG_RTK1), glioblastoma, IDH-wildtype, RTK2 subtype (HGG_RTK2), diffuse pediatric-type high-grade glioma, RTK1 subtype (HGG_pedRTK1), diffuse pediatric-type high-grade glioma, RTK2 subtype (HGG_pedRTK2) and diffuse pediatric-type high-grade glioma, MYCN subtype (HGG_pedMYCN), supratentorial ependymoma, ZFTA-fused (EPN_ZFTA), supratentorial ependymoma, YAP1-fused (EPN_YAP), supratentorial ependymoma, NOS (EPN_ST), posterior fossa group A ependymoma (EPN_PFA), posterior fossa group B ependymoma (EPN_PFB), myxopapillary enpendymoma (EPN_MPE)

Fig.2

Copy-number profile derived from DNA methylation array data showing a homozygous deletion of CDKN2A/B as well as structural alterations affecting chromosome 9q and 10p around the NTRK2 and KIF5B loci (a). Visualization of the KIF5B::NTRK2 gene fusion confirmed by RNA sequencing (b). Circos plot of the different gene fusions detected in the series (lines link fusion gene partners according to chromosomal location; c)

Fig.3

Visualization of the ATRX mutation profile in the investigated cohort was created using the online tool ProteinPaint available at https://proteinpaint.stjude.org/ (a). Immunohistochemical loss of ATRX expression in the tumor cells of case #01 and #18 (b, c). Scale bars 200 µm

Fig. 4

Typical histological features of tumors within this series showing tumor cells with inconspicuous nuclei and perinuclear clearing (a, b). Some cases show multinucleated cells (c, indicated by arrows). Cell density is variable (b, h). Tumors are densely vascularized (note the typical pattern, as f and i show two different cases). Microvascular proliferation is frequently observed (b, e). Necrosis is present in a subset of cases (d). An angiocentric arrangement was seen in single cases (g). Scale bars 200 µm

Fig. 5

Immunohistochemistry reveals diffuse positivity for synaptophysin with a weak or moderate cytoplasmic reactivity, while the tumor cell matrix shows a consistently strong staining (a). The majority of tumor cells shows negative or weak NeuN staining, although scattered positive cells are frequently present (b). Class III ß-tubulin (c) and NSE (d) are positive in the tumors, although sometimes restricted to a proportion of neoplastic cells. GFAP staining shows strong positivity of the tumor cell matrix with variable cytoplasmic reactivity between the cases and/or different areas within the same tumor (e). Olig2 staining is usually strongly positive (f). MAP2 staining is positive in all tumors (g). CD34 expression is restricted to the vessels (h). Ki-67 labeling index varies between the different cases, here showing a moderate proliferative activity (f). Scale bars 200 µm

Fig. 6

Clinical features of the investigated cohort. Age at diagnosis of the patients with a median age of 19 years (a), patient sex distribution (b) and distribution of tumor location (c). Progression-free (d) and overall survival (e) of the 18 patients from the investigated cohort for whom follow-up data were available. MRI (axial, T2) performed 12 months after surgery showing a small enhancing nodule along the cavity of resection (f). No sign of progression after 37 months of Larotrectinib (axial, T2; g)