Recurrent BCOR Internal Tandem Duplication and YWHAE-NUTM2B Fusions in Soft Tissue Undifferentiated Round Cell Sarcoma of Infancy: Overlapping Genetic Features With Clear Cell Sarcoma of Kidney

Abstract

Soft tissue undifferentiated round cell sarcoma (URCS) occurring in infants is a heterogenous group of tumors, often lacking known genetic abnormalities. On the basis of a t(10;17;14) karyotype in a pelvic URCS of a 4-month-old boy showing similar breakpoints with clear cell sarcoma of kidney (CCSK), we have investigated the possibility of shared genetic abnormalities in CCSK and soft tissue URCS. Most CCSKs are characterized by BCOR exon 16 internal tandem duplications (ITDs), whereas a smaller subset shows YWHAE-NUTM2B/E fusions. Because of overlapping clinicopathologic features, we have also investigated these genetic alterations in the so-called primitive myxoid mesenchymal tumor of infancy (PMMTI). Among the 22 infantile URCSs and 7 PMMTIs selected, RNA sequencing was performed in 5 and 2 cases, with frozen tissue, respectively. The remaining cases with archival material were tested for YWHAE-NUTM2B/E by fluorescence in situ hybridization (FISH) or reverse transcription-polymerase chain reaction (RT-PCR), and BCOR ITD by PCR. A control group of 4 CCSKs and 14 URCSs in older children or adults without known gene fusion and 20 other sarcomas with similar histomorphology or age at presentation were also tested. A YWHAE-NUTM2B fusion was confirmed in the index case by FISH and RT-PCR, whereas BCOR ITD was lacking. An identical YWHAE-NUTM2B fusion was found in another URCS case of a 5-month-old girl with a back lesion. The remaining cases and control group lacked YWHAE gene rearrangements; instead, consistent BCOR ITDs, similar to CCSK, were found in 15/29 (52%) infantile sarcoma cases (9/22 infantile URCS and 6/7 PMMTI). In the control cohort, BCOR ITD was found only in 3 CCSK cases but not in the other sarcomas. Histologically, URCS with both genotypes and PMMTI shared significant histologic overlap, with uniform small blue round cells with fine chromatin and indistinct nucleoli. A prominent capillary network similar to CCSK, rosette structures, and varying degree of myxoid change were occasionally seen. BCOR ITD-positive tumors occurred preferentially in the somatic soft tissue of the trunk, abdomen, and head and neck, sparing the extremities. RNAseq showed high BCOR mRNA levels in BCOR ITD-positive cases, compared with other URCSs. In summary, we report recurrent BCOR exon 16 ITD and YWHAE-NUTM2B fusions in half of infantile soft tissue URCS and most PMMTI cases, but not in other pediatric sarcomas. These findings suggest a significant overlap between infantile URCS and CCSK, such as age at presentation, histologic features, and genetic signature, thus raising the possibility of a soft tissue counterpart to CCSK.

Figure 1. The index URCS case with YWHAE-NUTM2B fusion

Diffuse sheets of monotonous round cells with fine chromatin, indistinct nucleoli, frequent apoptosis and occasional mitoses (A, H&E, 400X). Representative karyotype showing a three-way translocation t(10;17;14)(q22.1;p13.3;q24) indicated by the arrows (B). FISH showing YWHAE rearrangement, with break-apart green (telomeric) and red (centromeric) signals (C, white arrows). Sanger sequencing of RT-PCR product demonstrating YWHAE exon 5 fused to NUTM2B exon 2 (D).

Figure 2. Histology of soft tissue URCS with YWHAE-NUTM2B fusion (A) or BCOR ITD (B–F)

Second case with YWHAE-NUTM2B fusion showing delicate arborizing vascular network reminiscent to CCSK (A, URCS2, 200X). BCOR ITD URCS showing prominent capillary network (B, URCS3, 100X); cellular fibrous septa (C, URCS3, 200X); uniform round to ovoid tumor cells (B); extensive myxoid stroma and cystic spaces (D, URCS 6, 100X); rosette formation (E, URCS5, 200X) and vacuolated cytoplasm (F, URCS6, 400X).

Figure 3. Spectrum of BCOR ITD variants in URCS, PMMTI and CCSK samples

(A) Schematic diagram showing the location of BCOR on chromosome Xp11.4, protein domains of BCOR, the location of the duplicated sequences on PUFD domain, and the ITD subtypes among URCS, PMMTI and CCSK. Compared to wild-type BCOR, the original and duplicated sequences are shown in blue and red, respectively. The intervening sequences in black indicate insertions; however, all ITD variants are in-frame. The common region of duplicated sequence is indicated by vertical dot lines. BBD, BCL-6 binding domain; ANK, ankyrin repeat; PUFD, PCGF Ub-like fold discriminator. (B) By unsupervised clustering, the URCS/PMMTI with BCOR ITD (red), YWHAE-NUTM2B fusion (blue) and BCOR-MAML3 fusion (orange) grouped together in a distinct cluster from other sarcomas available on the RNAseq.

Figure 4. Morphologic spectrum of PMMTI

The PMMTI were associated with a monomorphic cytomorphology, ranging from round (A, PMMTI6, 400X), stellate (B, PMMTI6, 400X) to spindle (C, PMMTI2, 400X). Overlapping histologic features with BCOR ITD URCS and CCSK were noted, including rich vascular network (D, PMMTI6, 100X) and cellular fibrous septa (E, PMMTI3, 200X). Other features included occasional cytoplasmic vacuoles (F, PMMTI6, 400X) or rhabdoid cells (G, PMMTI, 400X). The only PMMTI lacking BCOR ITD showed ovoid to stellate tumor cells, with slightly clumped chromatin and myxoid stroma (H, PMMTI7, 400X).

Figure 5. URCS/PMMTI with BCOR ITD display overlapping gene signature with CCSK and BCOR-CCNB3 positive sarcoma

(A) Venn diagram showing overlapping differential gene signature between BCOR ITD URCS (RNA-seq data), CCSK (published data, Illumina HumanHT-12 V4.0 beadchip platform), and round cell sarcoma with BCOR-CCNB3 (published data, Affymetrix Human Genome U133A Plus 2 platform). The GSEA analysis demonstrates enrichment of the 44 genes in common among the 3 tumor categories. Subsequent supervised hierarchical clustering using this 44 gene list revealed that BCOR ITD-positive and YWHAE fusion-positive tumors grouped together, separate from the control groups. NES, normalized enrichment score; NOM P, nominal p-value; FDR, false discovery rate. (B) Comparative expression of commonly upregulated genes: BCOR, ZIC2, KDM2B, PITX1, LHX2 and SATB2, across these 3 tumor entities and individual platforms (URSC with BCOR ITD, YWHAE-NUTM2B, BCOR-MAML3 on RNAseq; BCOR ITD and YWHAE-NUTM2B/E CCSK on Illumina HumanHT-12 V4.0 beadchip platform; and round cell sarcoma with BCOR-CCNB3 on Affymetrix Human Genome U133A Plus 2 platform). ‘Other URCS’ indicates URCS without known genetic alterations.

Figure 6. Histologic overlap among CCSK and URCS with BCOR ITD and other BCOR-related fusions

A similar cytomorphology with fine chromatin and delicate vascular network in CCSK (A, 400X), BCOR-MAML3 URCS (B, 400X), and BCOR-CCNB3 URCS (C, 400X).