Recurrent NCOA2 gene rearrangements in congenital/infantile spindle cell rhabdomyosarcoma

Abstract

Spindle cell rhabdomyosarcoma (RMS) is a rare form of RMS with different clinical characteristics between children and adult patients. Its genetic hallmark remains unknown and it remains debatable if there is pathogenetic relationship between the spindle cell and the so-called sclerosing RMS. We studied two pediatric and one adult spindle cell RMS by next generation RNA sequencing and FusionSeq data analysis to detect novel fusions. An SRF-NCOA2 fusion was detected in a spindle cell RMS from the posterior neck in a 7-month-old child. The fusion matched the tumor karyotype and was confirmed by FISH and RT-PCR, which showed fusion of SRF exon 6 to NCOA2 exon 12. Additional 14 spindle cell (from 8 children and 6 adults) and 4 sclerosing (from 2 children and 2 adults) RMS were tested by FISH for the presence of abnormalities in NCOA2, SRF, as well as for PAX3 and NCOA1. NCOA2 rearrangements were found in two additional spindle cell RMS from a 3-month-old and a 4-week-old child. In the latter tumor, TEAD1 was identified by rapid amplification of cDNA ends (RACE) to be the NCOA2 gene fusion partner. None of the adult tumors were positive for NCOA2 rearrangement. Despite similar histomorphology in adults and young children, these results suggest that spindle cell RMS is a heterogeneous disease genetically as well as clinically. Our findings also support a relationship between NCOA2-rearranged spindle cell RMS occurring in young childhood and the so-called congenital RMS, which often displays rearrangements at 8q13 locus (NCOA2).

Figure 1. Spindle cell rhabdomyosarcoma with SRF-NCOA2 fusion

This tumor is from a 7-month old child who presented with a posterior neck mass (case RMS2). (A) It is composed of monotonous spindle cells arranged in long fascicles, resembling leiomyosarcoma. The tumor infiltrates the adjacent fibroadipose tissue (original magnification 100×). By immunohistochemistry, it is diffusely positive for desmin (B) and multifocal nuclear positivity for myogenin (C). (D) Schematic representation of SRF-NCOA2 fusion involving exons 12 and 6, respectively. The numbers indicate exons. (E) This result was confirmed by RT-PCR which identified an in frame fusion of the SRF exon 6 with exon 12 of NCOA2, by direct sequencing of the 705 bp amplified product (F). FISH assay demonstrated break-apart signals for both SRF (G) and NCOA2 (H) (Probes centromeric to SRF and NCOA2 are in red, probes telomeric to SRF and NCOA2 are in green). (I) Number of reads normalized per million mapped nucleotides demonstrated differential expression of NCOA2 5′ and 3′ to the breakpoint, which supports the orientation of fusion transcript.

Figure 2. Histomorphology of NCOA2-negative spindle cell and sclerosing rhabdomyosarcoma

A variegated histological pattern is observed in spindle cell rhabdomyosarcoma including (A) smooth muscle-like appearance (case RMS3), (B) fibrosarcoma-like features (case RMS9), (C) occasionally mimicking MPNST (case RMS8) and (D) a hypocellular, densely collagenized background in a sclerosing RMS (RMS18). (A, original magnification 100×; B-C, original magnification 200×).

Figure 3. Spindle cell rhabdomyosarcoma with TEAD1-NCOA2 fusion

Tumor from a 4 week-old baby boy who was born with a chest wall mass (case RMS4). (A) It is composed of short fascicles of spindle cells that have somewhat myofibroblastic appearance, infiltrating adjacent skeletal muscle. (B) A more cellular high grade component was also noted arranged in intersecting fascicles and associated with a high mitotic activity. Tumor cells demonstrate patchy positivity for Desmin and focal nuclear reactivity for myogenin (C) (A-C, original magnification 200×). (D) Partial karyotype showing a t(8;11) translocation. (E) Direct sequencing of the RT-PCR product confirmed the fusion of the TEAD1 exon 8 with NCOA2 exon 13. (F) FISH assay demonstrated TEAD1 and NCOA2 fusion-signal (highlighted with blue arrows; probes centromeric to NCOA2 in red, probes telomeric to TEAD1 in green).