Report two adult cases of high-grade neuroepithelial neoplasm harbouring EP300::BCOR fusions with comprehensive molecular detection.

Central nervous system (CNS) tumours with BCOR (BCL-6 transcriptional corepressor) internal tandem duplication (ITD) have emerged as a new molecular entity in recent years [1]. This was first reported by Sturm et al. in a study of 323 patients with CNS tumours, where this type of tumour was named ‘high-grade neuroepithelial tumour with BCOR ITD’ (HGNET BCOR ITD) [2]. In the fifth edition of the WHO classification of CNS tumours, it is now defined as ‘a malignant CNS tumour characterized by a predominantly solid growth pattern, uniform oval or spindle-shaped cells with a dense capillary network and focal pseudorosette formation, and an ITD in exon 15 of the BCOR gene’. However, the complete clinicopathological and molecular features, as well as the ideal treatment and prognosis, have not been fully elucidated. Furthermore, in addition to BCOR-ITD, other BCOR gene variants, such as EP300::BCOR [3, 4] and BCOR::CREBBP [5] fusion, have recently been reported, broadening the genetic alteration spectrum for this entity. To date, this tumour has been mainly reported in children and infants, cases in adults are rarely reported and less understood. However, we have recently encountered two cases of adult HGNET harbouring the fusion of EP300::BCOR. Clinical, histopathological and immunohistochemical (IHC) features of both cases were investigated. Comprehensive molecular analyses were conducted, including methylation profiling, Sanger sequencing, targeted next-generation sequencing (NGS) and reverse transcription-polymerase chain reaction (RT-PCR). Both cases exhibited similar imaging and pathological characteristics, and in addition to BCOR gene fusions, deep sequencing revealed other genetic variations that may have significant clinical implications for HGNETs with BCOR fusions. The first patient (Case 1), a previously healthy, 54-year-old male, was asymptomatic and discovered to have a mass in the right frontal lobe during a physical examination 5 days prior. Magnetic resonance imaging (MRI) revealed a large mass with clear boundaries in the right frontal lobe, showing nodular enhancement signals within the tumour and a shift towards the left side of the midline(Figure 1A). Case 1 underwent a total resection, followed by radiation therapy (6000 cGy) and adjuvant temozolomide therapy. At the 16-month follow-up, there were no signs of tumour recurrence. The pathological examination of Case 1 revealed a predominantly ependymoma-like pattern, characterized by abundant perivascular pseudorosettes, where the tumour cells formed a nuclear-free zone around the blood vessels (Figure 1B). The tumour had a well-circumscribed border with sharp demarcation from the adjacent brain tissue. In some areas, short-fascicular or ribbon patterns were observed, and extensive calcified foci were also present (Figure 1D,E). The tumour exhibited high cellularity, nuclear atypia and increased mitosis in some regions (Figure 1C). Immunohistochemical staining showed that the tumour cells were mostly negative for GFAP and S-100, but focally positive for Olig2. Epithelial membrane antigen (EMA) staining revealed faint granular cytoplasmic stain, and Ki-67 was focally up to 30%. Other immunohistochemical markers showed that the tumour cells were diffusely positive for vimentin, CD56 and EGFR; partially positive for SATB2; sporadically positive for NeuN; and negative for BCOR, IDH1R132H and BRAF V600E (VE1) (Figure 1F,G). It was difficult to get an exact diagnosis based on the above results, so a methylation profiling was performed using the EPIC 850k BeadChip array (Illumina, San Diego, CA). By comparing the methylation profile with the selected reference cases from the published cohort (GSE109381) of 2801 previously published CNS tumours [6], this sample was invariably clustered together with the reference methylation class ‘CNS tumour with BCOR internal tandem duplication’ by unsupervised hierarchical clustering analysis (Figure 1H) and t-SNE analysis (Figure 1I). Methylation-based copy number profiling revealed gains in chromosomes 1q, 5 and 7; more importantly, we observed the chromosome 22q12.31 deletion, where EP300 is located, as previously described by Aldape et al. [7] (Figure 1J). In this case, however, Sanger sequencing did not identify any BCOR-ITD at exon 15. Several months later, we encountered the second patient (Case 2), a 43-year-old female who had been experiencing headaches for 10 days. Her headaches were associated with changes in posture, with pain occurring when lying flat but not when standing. MRI revealed a large, nodular demarcated and heterogeneous mass in the right frontotemporal lobe with an obvious mass effect (Figure 2A). Near-total resection of the lesion was achieved, and the patient received radiation therapy (5200 cGy) supplemented with adjuvant chemotherapy postoperatively. The patient had no signs of tumour progression at the 8-month follow-up. Histologically, Case 2 exhibited diverse appearances with a higher cellularity than Case 1. In some areas, the tumour cells were spindle or ovoid and densely arranged in sheets with branching capillary network (Figure 2B). The local area showed a high-grade ependymoma-like structure dominated by pseudo-rosettes, as well as Homer Wright rosettes with brisk mitosis (Figure 2C). Palisading necrosis was easily observed (Figure 2D), and microcyst structures and calcifications were frequently present (Figure 2E). Immunohistochemical analysis revealed that the tumour cells were diffusely and strongly positive for vimentin, CD56 and SATB2, weakly positive for BCOR, and focally positive for NeuN and Olig2 (Figure 2F–I). In contrast, staining for GFAP and S-100 was completely negative in Case 2. However, the amount of tumour tissue from Case 2 was insufficient for methylation testing after NGS detection. Targeted NGS results: the DNA-based and RNA-based NGS analyses were used to profile the two samples. In Case 1, the EP300::BCOR fusion was identified at both DNA and RNA levels with breakpoints located in exon 31 of EP300 and exon 6 of BCOR. Additionally, Case 1 showed a BCOR::L3MBTL2 fusion only at the RNA level with breakpoints in exon 4 of BCOR and exon 2 of L3MBTL2. No other genetic alterations were identified in this case. In Case 2, the EP300::BCOR fusion was also identified at both DNA and RNA levels with breakpoints in exon 31 of EP300 and exon 4 of BCOR. Furthermore, additional molecular alterations in Case 2 included a TP53 p.R273C missense mutation, a NOTCH3 p.G37E point mutation and PDGFRA amplification at the DNA level, as well as a PDGFRA (exon 9) and FIP1L1 (exon 4) fusion at the RNA level. The graphical depiction of the BCOR fusion products for both cases is shown in Figures 1K,I and 2J. RT-PCR was performed to further validate all BCOR fusion transcripts mentioned above. Agarose electrophoresis and Sanger sequencing of the amplified products confirmed the presence of the breakpoints detected by RNA-seq (Figures 1M and 2K). No BCOR-ITD was detected in either sample. The two adult cases exhibited comparable radiological and pathologic characteristics that are consistent with those of CNS HGNET-BCOR tumours documented in the literature. Many cases reported in the literatures were located supratentorialy and showed diverse histologic features including ependymoma-like, oligodendroglioma-like or glioblastoma-like [3, 7]. They often demonstrated a low-grade morphology with high-grade transformation [4]. The IHC characteristics of BCOR-fusion tumours were variable. Tumour cells were reported to be immunopositive for Olig2, GFAP, NeuN, EMA, EGFR or beta-catenin, possibly suggesting a potential for multidiretional differentiation [3-5]. Although BCOR IHC staining can be helpful, it often does not correspond with genetic tests. Therefore, methylation profiling combined with NGS detection could significantly enhance the diagnostic accuracy. Recently, Aldape et al. published a retrospective study of gliomas with EP300 and BCOR gene fusions [7]. Their study profiled 21 patients with CNS tumours harbouring BCOR fusions (11 with the EP300 gene, 10 with other or undetermined genes). The median age was 30 years, and 71% of the tumours were well-defined (10 were supratentorial and 5 were in the posterior fossa). Recurring histologic appearances included perivascular pseudorosettes, myxoid changes, calcifications or branching capillary networks. The authors noted a partially overlapping methylation profile between the BCOR-fusion group and the ITD group. They also reported that the median PFS for BCOR-fusion tumours was 45 months, which is favourable compared to the BCOR-ITD tumours [8]. Our two reported cases provide additional evidence for this type of tumour, which may have been underestimated in adults previously. According to our NGS detecting results, besides of fusions of EP300::BCOR, Case 1 also showed a fusion of BCOR::L3MBTL2, which has been previously reported as a confirmed somatic mutation in a 24-year-old female patient diagnosed with high-grade IDH-wild-type glioma [5]. Simultaneous detection of BCOR::L3MBTL2 and EP300::BCOR fusion was also reported in a female patient with uterine sarcoma, which showed moderate nuclear atypia, active mitosis and abundant vascular components [9]. The fusion of FIP1L1::PDGFRA in Case 2 may hold significance for targeted therapy. Previous studies have shown that the FIP1L1-PDGFRA fusion gene is a crucial oncogenic driver of chronic eosinophilic leukemia, which can be effectively targeted by tyrosine kinase inhibitors like imatinib, sunitinib or sorafenib [10]. Treatment with these drugs has resulted in rapid complete remission for patients. Therefore, if the tumour in Case 2 recurs in the future, drugs such as imatinib could be considered as a potential treatment option. In conclusion, BCOR fusion tumours may be distinguished from BCOR-ITD tumours of the CNS by older age, greater glial differentiation, an ependymoma-like appearance and a higher likelihood of a better prognosis. Further studies of additional cases are strongly needed to precisely characterize the classification of CNS tumours with BCOR alterations. With the help of effective molecular assays such as methylation profiling and NGS analysis, an increasing number of adult BCOR fusion tumours may be detected. Data sharing is not applicable to this article as no new data were created or analyzed in this study.