BCOR-CCNB3 Fusion Positive Sarcomas: A Clinicopathologic and Molecular Analysis of 36 Cases With Comparison to Morphologic Spectrum and Clinical Behavior of Other Round Cell Sarcomas

Abstract

BCOR-CCNB3 sarcoma (BCS) is a recently defined genetic entity among undifferentiated round cell sarcomas, which was initially classified as and treated similarly to the Ewing sarcoma (ES) family of tumors. In contrast to ES, BCS shows consistent BCOR overexpression, and preliminary evidence suggests that these tumors share morphologic features with other tumors harboring BCOR genetic alterations, including BCOR internal tandem duplication (ITD) and BCOR-MAML3. To further investigate the pathologic features, clinical behavior, and their relationship to other round cell sarcomas, we collected 36 molecularly confirmed BCSs for a detailed histologic and immunohistochemical analysis. Four of the cases were also analyzed by RNA sequencing (RNAseq). An additional case with BCOR overexpression but negative CCNB3 abnormality showed a novel KMT2D-BCOR fusion by targeted RNAseq. The patients ranged in age from 2 to 44 years old (mean and median, 15), with striking male predominance (M:F=31:5). The tumor locations were slightly more common in bone (n=20) than soft tissue (n=14), with rare visceral (kidney, n=2) involvement. Histologically, BCS showed a spectrum of round to spindle cells with variable cellularity, monomorphic nuclei and fine chromatin pattern, delicate capillary network, and varying amounts of myxoid or collagenous stroma. The morphologic features and immunoprofile showed considerable overlap with other round cell sarcomas with BCOR oncogenic upregulation, that is, BCOR-MAML3 and BCOR ITD. Follow-up available in 22 patients showed a 5-year overall survival of 72%, which was relatively similar to ES (79%, P=0.738) and significantly better than CIC-DUX4 sarcomas (43%, P=0.005) control groups. Local recurrences occurred in 6 patients and distant metastases (lung, soft tissue/bone, pancreas) in 4. Seven of 9 cases treated with an ES chemotherapy regimen with evaluable histologic response showed >60% necrosis in posttherapy resections. Unsupervised clustering by RNAseq data revealed that tumors with BCOR genetic alterations, including BCOR-CCNB3, BCOR-MAML3, and BCOR ITD, formed a tight genomic group distinct from ES and CIC-rearranged sarcomas.

Figure 1. Histologic spectrum of BCS with round to spindle cells and occasional myxoid stroma

(A) A predominant round cell morphology with uniform nuclei, fine chromatin pattern, and delicate vascular network; (B) Monomorphic spindle cell morphology arranged in a fascicular pattern, reminiscent of SS; (C) Short spindle to ovoid cells with vague whorling pattern; (D) Alternating hypercellular and hypocellular areas, mimicking MPNST. (E) Small amount of myxoid stroma between loosely arranged tumor cells is a common finding. (F–H) Less common features includes: cell clustering (F), cord-like arrangement (G), and microcystic formation (H); (I) Sharp contrast of hypercellular round cell component and hypocellular myxoid area

Figure 2. Infrequent morphologic patterns of BCS

(A) Ropey collagen fibers separating spindle tumor cells. (B) A more homogenous, silky collagenous background with scattered short spindle cells. (C) Osteoid matrix deposition in a metastatic lung lesion, but not in the primary tumor. (D–F) A chest wall tumor of a 15 year-old male shows alternating areas of hypocellular collagenous background (D, left upper; E) and hypercellular round cell component (D, right lower; F)(case 23). (G–I) A foot mass of a 14 year-old male shows a purely spindle cell tumor with low to intermediate cellularity (G) and thick hyalinized collagen (H) in the primary lesion (case 20). Local recurrences 1 year and 3 years later showed a predominant round cell morphology (I). Both the primary and recurrent specimens were positive for BCOR-CCNB3 fusion by FISH. BCOR IHC showed patchy moderate to strong staining in the primary lesion (H, inset).

Figure 3. FISH and RT-PCR testing for BCOR-CCNB3 fusions

(A) Normal signal pattern: green and yellow signal probes flanking telomeric and centromeric ends of BCOR, respectively, while red signals flank CCNB3 on each side. (B) Abnormal FISH pattern: green and yellow signals show break-apart, while red signals split into two signals, representing centromeric and telomeric CCNB3 signals. A BCOR-CCNB3 fusion is confirmed when the yellow (centromeric BCOR, 5’) comes together with one of the red signals. (C) Sanger sequencing of RT-PCR product showed BCOR exon 15 fused to CCNB3 exon 5.

Figure 4. ‘BCOR family of tumor’

(A) Schematic diagram showing BCOR-CCNB3, BCOR-MAML3 fusions, and BCOR ITD (common duplicated region underlined) involving the last exon (exon 15) of BCOR. Representative sequences from RNAseq data demonstrating the alterations of BCOR (in blue) only a few nucleotides away from each other. (B) At exon level, up-regulations of BCOR in BCOR-CCNB3 (red) and BCOR ITD (orange) are observed up to the last exon, consistent with the genetic change at the end of exon 15. Black dots indicate other soft tissue tumors in the same platform as control. (C) Unsupervised clustering using RNAseq data shows clustering of BCOR-CCNB3 sample (red) with BCOR ITD (orange) and BCOR-MAML3 (purple) into a group, separate from Ewing sarcomas (blue) and CIC-rearranged sarcomas (green).

Figure 5. Immunohistochemistry of BCOR-CCNB3 tumors

(A) BCOR staining is typically diffuse with a strong nuclear pattern. (B) Post-chemotherapy resection of case 7 showing 90% tumor necrosis and foci of residual viable tumor cells (inset) with retained BCOR staining. SATB2 (C), TLE1 (D), and cyclin D1 (E) are also expressed in the majority of cases.

Figure 6

(A) Venn diagram showed limited transcriptional overlap between BCS, ES, and SS gene (11 genes in common). Additionally, 64 genes were shared between BCS and SS, and 8 genes between BCS and ES. (B) Supervised clustering using the BCS gene signature showed that ES (green), BCS (red), and most SS (blue) samples clustered into distinct individual clusters.

Figure 7

Overall survival of 22 BCS (blue), 121 ES (black), 34 SS (green), and 57 CIC-rearranged sarcomas (red). BCS was associated with a more favorable outcome compared to CIC-rearranged sarcoma (p=0.005), while no significant survival difference was noted between BCS and ES (p=0.738) or BCS and SS (p=0.802). Duration is shown in months.