Mutation profiling of anaplastic ependymoma grade III by Ion Proton next generation DNA sequencing

Abstract

Background: Ependymomas are glial tumors derived from differentiated ependymal cells. In contrast to other types of brain tumors, histological grading is not a good prognostic marker for these tumors. In order to determine genomic changes in an anaplastic ependymoma, we analyzed its mutation patterns by next generation sequencing (NGS). Methods: Tumor DNA was sequenced using an Ion PI v3 chip on Ion Proton instrument and the data were analyzed by Ion Reporter 5.6. Results: NGS analysis identified 19 variants, of which four were previously reported missense variants; c.395G>A in IDH1, c.1173A>G in PIK3CA, c.1416A>T in KDR and c.215C>G in TP53. The frequencies of the three missense mutations ( PIK3CA c.1173A>G, KDR c.1416A>T, TP53, c.215C>G) were high, suggesting that these are germline variants, whereas the IDH1 variant frequency was low (4.81%). However, based on its FATHMM score of 0.94, only the IDH1 variant is pathogenic; other variants TP53, PIK3CA and KDR had FATHMM scores of 0.22, 0.56 and 0.07, respectively. Eight synonymous mutations were found in FGFR3, PDGFRA, EGFR, RET, HRAS, FLT3, APC and SMAD4 genes. The mutation in FLT3 p.(Val592Val) was the only novel variant found. Additionally, two known intronic variants in KDR were found and intronic variants were also found in ERBB4 and PIK3CA. A known splice site mutation at an acceptor site in FLT3, a 3'-UTR variant in the CSF1R gene and a 5'_UTR variant in the SMARCB1 gene were also identified. The p-values were below 0.00001 for all variants and the average coverage for all variants was around 2000x. Conclusions: In this grade III ependymoma, one novel synonymous mutation and one deleterious missense mutation is reported. Many of the variants reported here have not been detected in ependymal tumors by NGS analysis previously and we therefore report these variants in brain tissue for the first time.

Keywords: Anaplastic Ependymoma; Ependymoma; Glioma; Ion Proton; Next Generation DNA sequencing; Palisading necrosis; Perivascular psuedorossettes; pediatric brain tumors.

Figure 1.. Grade III ependymoma unenhanced computed tomography (CT) of the brain.

A large lesion (5.4 × 7.5cm) in the left cerebral frontoparietal location with predominantly cystic components (panel A, green arrow), and a large, eccentric clump of coarse calcification (panel B; yellow arrow). Mass effect and mid line shift (panel C, red arrow) can also be seen. No hydrocephalic changes or intrinsic active hemorrhagic focus were observed.

Figure 2.. Hematoxylin and eosin (H&E) staining showing anaplastic ependymoma features.

( A) Focal calcification areas (blue arrow), and perivascular pseudo-rosettes (white arrow). ( B) Pseudo palisading necrosis, characterized by a garland-like structure of hypercellular tumor nuclei (black arrow) lining up around irregular foci of tumor necrosis (blue arrow). ( C) The cellular tumor exhibiting glomeruloid vascular proliferation (black arrows). ( D) Extensive palisading necrosis (green arrows) and true rosettes (yellow arrows).

Figure 3.. Hematoxylin and eosin (H&E) staining showing anaplastic ependymoma features.

( A) Pseudo palisading necrotic areas, exhibiting true rosettes with central lumen (yellow arrow). ( B) Focal areas with numerous tumor giant cells and the presence of a brisk mitotic activity (green arrows). ( C) Tumor with vascular formation (yellow arrows) and pseudo palisading necrotic areas. ( D) Formation of true rosettes (green arrows) surrounding the microvascular proliferation within ependymal tumors, usually signifies anaplastic transformation which is characteristic of ependymomas.

Figure 4.. Photomicrographs of Ki-67, vimentin, GFAP, and EMA immunostaining of the ependymal tumor.

( A) Ki-67 immunostaining indicates a high proliferation index in the tumor (70%). ( B) Vimentin stain is positive. ( C) GFAP stain is positive. ( D) EMA stain is positive and shows punctate cytoplasmic (perinuclear dot-like) staining, fairly diagnostic of the ependymal nature of the tumor cells.

Figure 5.. Photomicrographs of beta-Catenin and E-Cadherin immunostaining of the ependymal tumor.

Immunostaining is strongly positive for beta-Catenin (panel A 20x, panel B 40x) and true rosettes (red arrows) and palisading cells (blue arrow) are clearly visible. E-Cadherin stain is also positive in this tumor. Red and blue arrows indicate tumor cells arranged in true rossettes and formation of palisading structures, respectively (panel C 20x, panel D 40x).

Figure 6.. iVariant analysis of variant characteristics.

Distribution of variants according to filters, showing characteristics including the relative number of variants located on each chromosome, variant class, substitution type and the functional consequences of each variant, in order to interpret and score the severity and impact of variants and therefore predict the severity of the disease. Doughnut charts in panels shows variants passed for each individual filter for ( A) Chromosomal distribution, ( B) Region in the gene, ( C) Variant class, ( D) Variant effect on the protein structure, ( E) Variant impact on the protein function and ( F) Clinical significance of the variants as annotated on the ClinVar database.

Figure 7.. Heat Map showing variant impact of each gene detected in the ependymal tumor.

Variant impact takes into account the type of mutation (such as insertion, deletion or frame shift) and considers the location of the variant (intronic or exonic). The color gradation from green to red indicates unknown, synonymous, missense, nonsense and splice variants, calculated based upon their SIFT, PolyPhen2 and Grantham scores.