Role of Immunohistochemistry in the Identification of Supratentorial C11ORF95-RELA Fused Ependymoma in Routine Neuropathology

Abstract

Ependymomas (EPs) are tumors of the brain and spinal cord constituting ∼10% of the childhood central nervous system neoplasms and about 30% in children aged <3 years. Their anatomic distribution varies according to the age, with those arising in the supratentorial (ST) compartment, spinal cord being more common in older children and adults, and those at the infratentorial location are more common and occurring more frequently in infants and children. Recently, molecular classification of EP subgroups has been proposed and a supratentorial ependymoma subgroup characterized by RELA-fusion genes (ST-EP-RELA) has been established. It would be useful to define a standardized, robust method for the diagnosis of these relevant fusion genes. We used real-time polymerase chain reaction, conventional real-time polymerase chain reaction, and Sanger sequencing to characterize RELA fusion status in formalin-fixed paraffin-embedded samples from 42 ST-EPs (12 adults and 30 pediatric). We tested p65/RELA and L1CAM protein immunohistochemistry for their ability to predict RELA-fusion status. We reviewed clinical data to assess significant associations in this anatomic subgroup. Of the 42 patients, we identified RELA-fusion genes in 17 cases. L1CAM immunostaining displayed 94% sensitivity, 76% specificity, 73% positive predictive value (PPV), 95% negative predictive value (NPV). The p65/RELA immunostaining displayed 100% sensitivity, 92% specificity, 89.5% PPV, 100% NPV. Concordant double immunostaining improves PPV to 92.5% and maintains 100% NPV. Immunohistochemistry using both p65/RELA and L1CAM antibodies is valuable for ST-EP-RELA diagnosis: the negativity with both antibodies consistently predicts the absence of RELA fusions, whereas verification of fusion transcripts by molecular analyses is warranted only in single-positive or double-positive staining cases.

Childhood supratentorial ependymomas with YAP1‐MAMLD1 fusion: an entity with characteristic clinical, radiological, cytogenetic and histopathological features

“…At lower density, such structures frequently occur in other ependymoma variants and are believed to represent microlumina. As expected, none of the YAP1‐MAMLD1 fusion‐positive ependymomas showed a nuclear p65 RelA accumulation typical for NFkB‐activated, RELA fusion‐positive supratentorial ependymoma .…”

“…RELA fusions have been shown to be oncogenic by activation of the NFKB pathway. The tumors can be well detected by demonstration of the fusion or by immunohistochemistry for pathological accumulation of p65 RelA protein in the nucleus .…”

Diagnostics of pediatric supratentorial RELA ependymomas: integration of information from histopathology, genetics, DNA methylation and imaging

“…Previous studies showed that ependymomas carrying the C11orf95‐RELA fusion were characterized by a nuclear accumulation of p65‐RelA, indicating a pathological activation of the NFκB signaling pathway, and reported high correlation with genomic analyses . Recently, Gessi et al demonstrated the ability of p65‐RelA IHC to predict RELA fusion status compared to Sanger sequencing and real‐time polymerase chain reaction . However, the specificity of the p65‐RelA IHC positivity was not tested on other ependymomas and histological mimics.…”

Intracranial ependymomas: molecular insights and translation to treatment

“…For posterior fossa tumors, immunochemistry using NELL2 and LAMA2 or H3K27me3 antibodies has been shown to be fairly reliable in distinguishing between PF‐EPN‐A and PF‐EPN‐B tumors . The use of L1CAM and RelA/p65 antibodies have also been shown to be relatively accurate in identifying ST‐EPN‐RELA tumors . However, given the presence of a small number of false positives and negatives with these antibodies, whole genome DNA methylation analysis may still be required in the event significantly different treatments are recommended.…”