Clinically aggressive pediatric spinal ependymoma with novel MYC amplification demonstrates molecular and histopathologic similarity to newly described MYCN-amplified spinal ependymomas

Primary spinal cord tumors contribute to ≤ 10% of central nervous system tumors in individuals of pediatric or adolescent age. Among intramedullary tumors, spinal ependymomas make up ~ 30% of this rare tumor population. A twelve-year-old male presented with an intradural, extramedullary mass occupying the dorsal spinal canal from C6 through T2. Gross total resection and histopathology revealed a World Health Organization (WHO) grade 2 ependymoma. He recurred eleven months later with extension from C2 through T1-T2. Subtotal resection was achieved followed by focal proton beam irradiation and chemotherapy. Histopathology was consistent with WHO grade 3 ependymoma. Molecular profiling of the primary and recurrent tumors revealed a novel amplification of the MYC (8q24) gene, which was confirmed by fluorescence in situ hybridization studies. Although MYC amplification in spinal ependymoma is exceedingly rare, a newly described classification of spinal ependymoma harboring MYCN (2p24) amplification (SP-MYCN) has been defined by DNA methylation-array based profiling. These individuals typically present with a malignant progression and dismal outcomes, contrary to the universally excellent survival outcomes seen in other spinal ependymomas. DNA methylation array-based classification confidently classified this tumor as SP-MYCN ependymoma. Notably, among the cohort of 52 tumors comprising the SP-MYCN methylation class, none harbor MYC amplification, highlighting the rarity of this genomic amplification in spinal ependymoma. A literature review comparing our individual to reported SP-MYCN tumors (n = 26) revealed similarities in clinical, histopathologic, and molecular features. Thus, we provide evidence from a single case to support the inclusion of MYC amplified spinal ependymoma within the molecular subgroup of SP-MYCN. Supplementary Information The online version contains supplementary material available at 10.1186/s40478-021-01296-2.

Recently, a novel molecular subgroup of spinal EP with focal high-level MYCN (2p24) amplification was defined and found to be associated with dismal outcomes and malignant progression, despite aggressive management [20,41,43,49]. This molecular pathology will be newly recognized as a distinct subgroup of spinal cord EP (SP-MYCN) in the fifth edition of the WHO Classification of Tumors of the Central Nervous System [16,35]. Similar to SP-MP, SP-MYCN were found to develop in extramedullary spaces in sharp contrast to the intramedullary growth seen in SP-EP [20,41,49]. Herein, we report on an adolescent male with aggressive classic spinal EP harboring a novel focal amplification of the MYC oncogene located on chromosome 8q24. A 12-year-old male with no significant past medical history presented to the emergency room with a threemonth history of back pain and acute onset of weakness in the left lower extremity. Neurological examination was significant for left lower extremity weakness and ataxia. Magnetic resonance imaging (MRI) of the brain and spine revealed a localized avidly enhancing intradural, extramedullary mass occupying the dorsal spinal canal from C6 through T2. The tumor resulted in severe cord compression and mild edema (Fig. 1a, b). He underwent a gross total resection of the tumor followed by observation with serial imaging. Eleven months later, he re-presented with acute onset of lower extremity paresthesia and left-handed weakness. Spine MRI revealed tumor recurrence with further extension anteriorly and posterior to the cord from C2 through T1-T2 with resultant severe cord compression, again demonstrating avid enhancement (Fig. 1c, d). Management included subtotal resection of the recurrent mass, focal proton beam irradiation (50.4 Gy), followed by four cycles of chemotherapy with vincristine, etoposide, cyclophosphamide, and cisplatin, as per ACNS0831 [45], then oral vorinostat for seven months. The patient experienced further progression with distal metastases in the thoraco-lumbar region distal to his primary tumor, 16 months after first relapse (Fig. 1e, f ), and completed intensity-modulated radiation therapy (IMRT) of 36 Gy to the entire thecal sac. On his post-radiation MRI, there was evidence of progression of the cervical region tumor and the patient is therefore now undergoing focal re-irradiation of the cervical region tumor. A clinical summary is presented in Table 1.

Case presentation
Histopathologic examination of the initial resection specimen revealed a compact lobulated glial neoplasm with variable morphology. The majority of the tumor showed classic ependymoma features, including mildly pleomorphic tumor cells with round to oval nuclei arranged in perivascular pseudo-rosette (nuclear-free zone) formations, often surrounding capillaries showing microvascular proliferation (Fig. 2a). Other tumor areas showed clear cell change (Fig. 2b) or tanycytic morphology with elongate spindle cells. Densely cellular nests of mitotically active cells displaying conspicuous nucleoli were also present (Fig. 2c). Mitoses were counted at 3-4 per 10 high power fields (HPF). Squash preparations showed a branching angiocentric pattern of tumor cells (Additional File 1: Fig. S1a). Immunohistochemical stains demonstrated prominent perivascular glial fibrillary acidic protein (GFAP) staining (Fig. 2d) while Olig-2 nuclear staining was rare (Fig. 2e). Epithelial membrane antigen (EMA) showed perinuclear dot-like immunoreactivity (Fig. 2f ), while ring-like staining was much less frequent. The Ki-67 proliferation index was focally up to 20% (Additional File 1: Fig. S1b). A diagnosis of ependymoma, WHO grade 2, with histopathologic features bordering between WHO grade 2 and WHO grade 3. The subsequent recurrent/residual tumor specimen showed similar histologic features, with even more and molecular features. Thus, we provide evidence from a single case to support the inclusion of MYC amplified spinal ependymoma within the molecular subgroup of SP-MYCN. To further characterize the tumor, the patient was consented on an Institutional Review Board-approved translational research protocol and underwent comprehensive molecular profiling, including paired tumor/normal enhanced exome sequencing (eES) and RNA-sequencing (Additional File 1: Materials and Methods). This analysis included an evaluation of small single nucleotide variants (SNV), small insertion-deletions, copy number alterations (CNA), gene fusions, and aberrant gene expression. We sequenced disease-involved tissue from both the primary spinal cord tumor and localized tumor recurrence occurring 11 months after the primary tumor. We did not identify any cancer-or disease-associated SNVs or CNAs from the germline comparator peripheral blood. In addition, we did not identify any clearly medically meaningful somatic SNVs, small indels, or gene fusions. In both analyzed timepoints, the CNA profile was notable for a focal amplification of the MYC gene on 8q24 (Fig. 3a) and biallelic loss of 17p, including TP53 and likely consistent with an isochromosome 17q (Fig. 3b, Additional File 1: Table S1). Other CNA described from eES included segmental biallelic losses of 8q, 10q (including PTEN), and 19q (Additional File 1: Fig. S2, Additional File 1: Table S1). The University of California Santa Cruz (UCSC) Treehouse Initiative (https:// treeh ouseg enomi cs. ucsc. edu/ explo re-our-data/) is a collaborative data  sharing initiative whereby RNA-sequencing data from a breadth of tumor types are publicly available. We utilized a cohort of pediatric and adolescent/young adult central and peripheral nervous system tumors (n = 563) from the UCSC Treehouse Initiative to compare MYC gene expression. Consistent with the identified gene amplification, MYC was found to be overexpressed in both primary (log2 fold change: 3.95, P = 0.0007) and recurrent (log2 fold change: 4.57, P = 9.57 × 10 -5 ) tumors. Visualization of MYC expression for our described patient case relative to ependymoma (n = 41), glioma (n = 300), medulloblastoma (n = 129), and neuroblastoma (n = 199) patients from the UCSC Treehouse Initiative and our internal cohort confirmed this overexpression (Fig. 3c). Fluorescence in situ hybridization (FISH) of the MYC (8q24) and MYCN (2p24) loci was performed from formalin-fixed paraffin-embedded tissue to confirm the presence of MYC amplification. Greater than 20 copies of MYC were detected relative to a 2-copy state for the cen-  fossa group B)). Unsupervised clustering with a reference cohort of 501 methylation profiles spanning all 10 established molecular EPN groups clearly assigned the tumor from our described patient case to the SP-MYCN group (Fig. 4a) [20,38]. This result was confirmed in a repeated clustering restricted to reference cases of the four molecular spinal EPN groups (n = 66, Fig. 4b). Analyses of CNA plots of 52 spinal tumors predicted as SP-MYCN revealed a focal amplification of MYCN in 50/52 tumors, but no additional case with MYC amplification (Fig. 4c). Furthermore, when clustered with a cohort of ~ 80,000 DNA methylation profiles covering the entire spectrum of existing molecular CNS tumor classes, the described 3) of the classifier assigned the described patient case to the SP-MYCN group with a calibrated score of 0.99, which is above the cut-off for confident class prediction (0.9) [10], and thus confirms the assignment of the case to the molecular group of SP-MYCN.

Discussion and conclusions
This report describes a novel case of an aggressive recurrent progressive spinal cord ependymoma with histologic features of an anaplastic ependymoma harboring focal MYC amplification. Interestingly, DNA methylationbased classification assigned this case to the molecular group of SP-MYCN. MYC (8q24) encodes the c-MYC protein, a transcription factor that interacts with other proteins to regulate gene expression, including those that promote cell growth and proliferation [25,48]. Deregulation of MYC has been shown to stimulate and maintain tumorigenesis in ex vivo models [28,48]. MYC alterations are recurrently described amongst many different types of neoplasms, including pediatric brain tumors [6,25], with amplification being most frequently reported [25,28]. Glial and non-glial brain tumors harboring MYC amplification demonstrated a significantly worse prognosis [8,22,27,30,37,42,54]. Despite the high frequency of MYC gene alteration in human cancers, it has been rarely reported in ependymoma. A single individual was reported with a recurrent anaplastic ependymoma harboring an abnormal karyotype 46,XX,der(8)t(8;11)(q24;p11), − 11,add(?) t(?;11)(?;q13)) and MYC overexpression [13]. Notably, this tumor was not located in the spine but rather in the supratentorial region of the brain. Despite MYC overexpression, no evidence of MYC gene rearrangements nor amplification were identified [13]. Given the paucity of literature describing MYC alterations in ependymoma, we performed a literature review describing the clinical, histologic, and molecular features of the 26 reported spinal ependymomas with MYCN (2p24) amplification (Table 1 and Additional File 1: Table S1) [20,41,43,49]. Similar to our described patient case, the SP-MYCN tumors had distinct growth patterns, and typically arose intradurally and extramedullary with invasion of the spinal cord. Most of the SP-MYCN tumors were located in the cervical or thoracic spine and were commonly associated with nodular metastatic spread and diffuse leptomeningeal involvement [20,41,43,49]. Compared to other spinal ependymomas, SP-MYCN tumors were associated with aggressive behavior and unfavorable outcomes, despite intensive multi-modal therapies [20,41]. In the described patient case, the histopathologic features including perivascular pseudorosettes, microvascular proliferation, densely cellular nests of mitotically active cells, prominent perivascular GFAP staining, sparse Olig-2 nuclear staining, a dotlike staining pattern with EMA, as well as increased Ki-67 proliferation indices were also described in prior  9:192 reported examples of the molecular group SP-MYCN [20,49]. Furthermore, loss of chromosome 10 may be a recurrent finding among this tumor subgroup, seen in 8/19 (42%) individuals with available copy number data (Additional File 1: Table S1). Our described patient case with MYC amplification also demonstrated segmental losses across chromosome 10q, including the tumor suppressor PTEN. Amplification of MYC in combination with disruption of PTEN has been shown in prostate cancer to contribute to aggressive disease and poor outcomes [23,29]. Larger studies to assess the association of MYC or MYCN amplification with chromosome 10 loss and patient outcomes may be warranted. In vitro and in vivo MYC inhibition has demonstrated tumor regression, across numerous tumor types [1,11,47]; nevertheless, direct inhibition of MYC is challenging. Thus, efforts have shifted towards targeting MYC transcriptional targets and regulatory domains [11,25,51,53], including the study of BET inhibitors [2,21], CDK inhibitors [5], mTOR inhibitors [26], Aurora A-kinase inhibitors [9,17] and CHK1 inhibitors [40]. Additionally, histone deacetylase (HDAC) inhibitors have been shown to impede MYC-amplified Group 3 medulloblastoma tumor growth in vitro [14,15,39,44]. Due to the focal MYC amplification, our patient received single-agent vorinostat, an oral HDAC inhibitor that is well tolerated in children with relapsed CNS tumors [19,24,31,50], following his first relapse. However, he presented with progressive disease after seven months.
In summary, we report a unique case of an adolescent male with an aggressive spinal ependymal tumor harboring focal MYC amplification. DNA array-based methylation profiling confidently classified this tumor as SP-MYCN, a recently described subgroup of spinal ependymoma. Our described patient case demonstrates clinical, histologic, and molecular overlap with the newly described SP-MYCN subgroup. Thereby, we provide evidence to support the inclusion of MYC amplified spinal ependymoma within the molecular subgroup of SP-MYCN. Testing for MYC or MYCN gene amplification may be warranted in newly diagnosed spinal tumors to aid in tumor characterization. Future strategies should focus on investigating the efficacy of indirect MYC-targeting strategies, introducing new possibilities for improving the prognosis in patients with SP-MYCN.
Additional file 1: Supplementary methods and data.