Embryonal tumor with abundant neuropil and true rosettes (ETANTR) is a recently recognized, rare embryonal CNS tumor, which predominantly occurs in young children and is associated with a highly aggressive disease course [1–3, 5–8, 13, 14, 18, 19, 23]. The histopathological diagnosis of ETANTR is based on the presence of primitive neuroectodermal tumor cells forming distinct multilayered ‘ependymoblastic’ rosettes and characteristic neuropil islands. Recently, genome-wide analyses have revealed a novel amplification at 19q13.42 [16, 19], which is meanwhile considered the genetic hallmark of ETANTR [13, 16]. The characteristic clinical, morphological, and genetic features support the concept of a distinct CNS PNET variant and suggest its introduction to the WHO classification of Tumors of the Central Nervous System . As the amplification at 19q13.42 has also been found in the vast majority of ependymoblastomas analyzed to date [13, 16], the common genetic background suggests the fusion of these two tumor types to a single entity.
Herein, we report for the first time the evolution of morphological features and genetic aberrations during the disease course in a patient with ETANTR. A 33-month-old girl presented with a 6-month history of episodic headaches, increased head circumference and mild gait disturbance. Magnetic resonance (MR) imaging showed a 9.6 × 8.6 × 11.7 cm left parieto-occipital, space-occupying, partly cystic lesion displaying T1-weighted hypo- to isointense signals (Fig. 1a) with cerebrospinal fluid-intense cysts on FLAIR sequence (Fig. 1b), and marked choline/creatine increase as a sign of cell proliferation on single voxel spectroscopy (Fig. 1c). Near-total surgical resection was performed. Histopathology revealed a primitive neuroectodermal tumor with highly cellular areas. Furthermore, hypocellular neuropil islands and multilayered rosettes were encountered, prompting the diagnosis of ETANTR (Fig. 2a–c). Immunohistochemistry demonstrated widespread expression of neuronal markers including synaptophysin and neurofilaments, EMA was detected solely within the rosettes in a dot-like and luminal surface pattern. SMARCB1/INI1 nuclear protein expression was retained. Fluorescence in situ hybridization (FISH) and a SNP array (Affymetrix SNP Array 6.0) demonstrated amplification at 19q13.42 (Fig. 3a, b). The SNP array revealed additional DNA copy number alterations including trisomies of chromosomes 2, 14, 17, 19, and 20, as well as partial gain of 1q (Fig. 3b). Postsurgical spinal imaging and cerebrospinal fluid cytology were negative. The child was treated according to an intensified therapy protocol (three cycles of a modified HIT 2000 SKK protocol  including vincristine, cyclophosphamide, methotrexate, etoposide, and ifosfamide, accompanied by intrathecal administration of etoposide and liposomal cytarabine) followed by high-dose chemotherapy according to a modified Finlay protocol  with stem cell rescue and cranial irradiation to a total of 54 Gy. After 10 months, the tumor was progressive with cerebrospinal fluid dissemination. Subtotal resection was performed. Histopathology showed a marked increase in tumor cell size, cellular pleomorphism with frequent nuclear moulding and wrapping, and prominent nucleoli indicating malignant progression towards a large cell/anaplastic phenotype (Fig. 2d). The small biopsy specimen contained neither areas of neuropil nor ependymoblastic rosettes. FISH and SNP array revealed still amplification at 19q13.42. Interestingly, FISH demonstrated a significant increase in the proportion of cells harboring the amplification, as well as condensation and fusion of the amplified signals (Fig. 3a). Compared to the primary tumor, the SNP array revealed balanced profiles of chromosomes 14, 17, 19, and 20, but loss of 17p, 18p and proximal 18q as well as a complex rearrangement of chromosome 19q were detectable (Fig. 3b). The child continued on an anti-angiogenic treatment (Peyrl et al., in press in Pediatric Blood & Cancer) that consisted of thalidomide, fenofibrate, celecoxib, etoposide, cyclophosphamide, and bevacizumab. Four months later the clinical condition deteriorated. The patient died 15 months after the initial diagnosis. Post-mortem examination of the brain revealed a left parietal resection cavity surrounded by soft greyish tumor masses, which extended to the contralateral hemisphere, covered the ventricular surfaces, and filled the basal cisterns. Upon histopathology, the large cell/anaplastic tumor cells showed a striking nucleolar enlargement, which consisted of ultrastructurally dense filamentous zones (Fig. 2e–h). However, despite extensive sampling procedures neither neuropil islands nor multilayered rosettes were detectable.
The clinical characteristics of this patient, including young age and unfavorable outcome despite intensified treatment, as well as anaplastic progression of the tumor tissue are in accordance with previous reports . Whereas loss of neuropil islands has been already observed in recurrences , the absence of multilayered rosettes has only been noticed in a single recurrent tumor biopsy so far . This case is unique, as we can exclude a sampling error through extensive autoptic investigation, and confirm the loss of all morphological key features of ETANTR during disease progression. Interestingly, the amplification at 19q13.42 was retained at relapse, and moreover a significant increase in the proportion of cells harboring the amplification was noted. The latter could correspond to the prominent nucleoli in the recurrent tumor tissue. Enlarged nucleoli are a common feature of many cancers [11, 22], and have been linked to malignant transformation  and cytotoxic treatment . In cell cultures of peripheral pediatric neuroblastoma, activated nucleoli indicate amplification of the MYCN gene . In pediatric medulloblastoma, prominent nucleoli are typically found in the large cell subtype and are frequently associated with MYCC or MYCN amplification  Neither MYCC nor MYCN amplification was noted in this case and MYCN amplification does not seem to play a role in ETANTR .
In the recurrent tumor tissue, additional genomic aberrations were found, such as loss of 17p including tumor suppressor TP53, which could play a role in anaplastic progression. The observed changes of genetic aberrations and morphological features support the concept of selection of a therapeutically resistant, more aggressive clone during cytotoxic treatment. Further paired genetic analyses could elucidate the mechanisms underlying the biological evolution of the tumor.
In the literature, single cases have been reported, which were initially diagnosed as central neurocytoma or medulloepithelioma, but exhibited characteristic histopathological features of ETANTR at relapse [2, 3]. This obviously variable morphology could lead to misdiagnosis of ETANTR. Accurate diagnosis of ETANTR is of high clinical relevance because of its poor response to current PNET treatment protocols and thus frequent fatal outcome. Therefore, genetic analysis of 19q13.42 contributes to diagnostic accuracy and should be performed in all CNS PNETs in children.