Novel BCOR-MAML3 and ZC3H7B-BCOR Gene Fusions in Undifferentiated Small Blue Round Cell Sarcomas

Abstract

Small blue round cell tumors (SBRCTs) are a heterogenous group of tumors that are difficult to diagnose because of overlapping morphologic, immunohistochemical, and clinical features. About two-thirds of EWSR1-negative SBRCTs are associated with CIC-DUX4-related fusions, whereas another small subset shows BCOR-CCNB3 X-chromosomal paracentric inversion. Applying paired-end RNA sequencing to an SBRCT index case of a 44-year-old man, we identified a novel BCOR-MAML3 chimeric fusion, which was validated by reverse transcription polymerase chain reaction and fluorescence in situ hybridization techniques. We then screened a total of 75 SBRCTs lacking EWSR1, FUS, SYT, CIC, and BCOR-CCNB3 abnormalities for BCOR break-apart probes by fluorescence in situ hybridization to detect potential recurrent BCOR gene rearrangements outside the typical X-chromosomal inversion. Indeed, 8/75 (11%) SBRCTs showed distinct BCOR gene rearrangements, with 2 cases each showing either a BCOR-MAML3 or the alternative ZC3H7B-BCOR fusion, whereas no fusion partner was detected in the remaining 4 cases. Gene expression of the BCOR-MAML3-positive index case showed a distinct transcriptional profile with upregulation of HOX-gene signature, compared with classic Ewing's sarcoma or CIC-DUX4-positive SBRCTs. The clinicopathologic features of the SBRCTs with alternative BCOR rearrangements were also compared with a group of BCOR-CCNB3 inversion-positive cases, combining 11 from our files with a meta-analysis of 42 published cases. The BCOR-CCNB3-positive tumors occurred preferentially in children and in bone, in contrast to alternative BCOR-rearranged SBRCTs, which presented in young adults, with a variable anatomic distribution. Furthermore, BCOR-rearranged tumors often displayed spindle cell areas, either well defined in intersecting fascicles or blending with the round cell component, which appears distinct from most other fusion-positive SBRCTs and shares histologic overlap with poorly differentiated synovial sarcoma.

Figure 1. Clinico-pathologic findings of the index SBRCT1 with BCOR-MAML3 fusion

Abdominal CT scan at presentation (A) showing a large intra-abdominal mass in the subphrenic/ subhepatic region, and after 19 months documenting rapid progression, with widespread metastases in the liver and peritoneum despite intensive chemotherapy and radiotherapy therapy (B,C). Morphologic appearance showing sheets of primitive round to ovoid cells set in a slightly collagenized stroma (D), with relatively uniform nuclei with fine stippled chromatin, eosinophilic scant cytoplasm, and a high mitotic rate (E); remained unchanged after chemo (F); with diffuse and strong membranous CD99 positivity (G).

Figure 2. BCOR-MAML3 gene fusion discovery and experimental validation (SBRCT1)

(A) Schematic representation of the BCOR-MAML3 fusion indicating the chromosomal loci and exonic structure of the candidate transcript; (B) Alignment of the RNA reads covering the fusion junction detected by Fusion Seq algorithm; (C) RT-PCR validation demonstrates the junction sequence spanning BCOR exon 15 and MAML3 exon 2; (D) Significant BCOR and MAML3 mRNA upregulation noted by RNA sequencing, compared to other translocation positive SBRCTs (left panels, including EWSR1-ERG, CIC-DUX4 fusions); as predicted from the structure of the chimeric BCOR-MAML3 fusion, exons 2–6 of MAML3 showed differential overexpression compared to MAML3 exon 1 in SBRCT1 compared to other translocation positive sarcomas (right panel).

Figure 3. FISH detection of BCOR-MAML3 fusion (SBRCT2)

Break-apart assay showing a split signal in BCOR (A) and in MAML3 (B) (A,B: green telomeric, red centromeric); Three-color fusion FISH assay showing co-localization of BCOR (red) with MAML3 (yellow, centromeric) and the MAML3 split of the green (telomeric) and yellow (centromeric) (C).

Figure 4. Morphologic spectrum of BCOR rearranged SBRCTs

Areas of spindling arranged in long fascicles or herring-bone pattern, which at high power shows ill-defined cell borders, ovoid nuclei with stippled chromatin (A,B: SBRCT2, BCOR-MAML3-positive); geographic areas of necrosis and areas of spindled morphology (C,D: SBRCT4, ZC3H7B- BCOR fusion). Monomorphic proliferation of round to oval cells showing a vague streaming pattern in a predominantly myxoid background (E, SBRCT7); primitive round cell morphology and geographic necrosis (F, SBRCT8).