Novel MIR143-NOTCH fusions in benign and malignant glomus tumors

Abstract

Glomus tumors (GT) have been classified among tumors of perivascular smooth muscle differentiation, together with myopericytoma, myofibroma/tosis, and angioleiomyoma, based on their morphologic overlap. However, no molecular studies have been carried out to date to investigate their genetic phenotype and to confirm their shared pathogenesis. RNA sequencing was performed in three index cases (GT1, malignant GT; GT2, benign GT and M1, multifocal myopericytoma), followed by FusionSeq data analysis, a modular computational tool developed to discover gene fusions from paired-end RNA-seq data. A gene fusion involving MIR143 in band 5q32 was identified in both GTs with either NOTCH2 in 1p13 in GT1 or NOTCH1 in 9q34 in GT2, but none in M1. After being validated by FISH and RT-PCR, these abnormalities were screened on 33 GTs, 6 myopericytomas, 9 myofibroma/toses, 18 angioleiomyomas and in a control group of 5 sino-nasal hemangiopericytomas. Overall NOTCH2 gene rearrangements were identified in 52% of GT, including all malignant cases and one NF1-related GT. No additional cases showed NOTCH1 rearrangement. As NOTCH3 shares similar functions with NOTCH2 in regulating vascular smooth muscle development, the study group was also investigated for abnormalities in this gene by FISH. Indeed, NOTCH3 rearrangements were identified in 9% of GTs, all present in benign soft tissue GT, one case being fused to MIR143. Only 1/18 angioleiomyomas showed NOTCH2 gene rearrangement, while all the myopericytomas and myofibroma/toses were negative. In summary, we describe novel NOTCH1-3 rearrangements in benign and malignant, visceral, and soft tissue GTs.

Fig. 1. MIR143-NOTCH2 gene fusion in a malignant gastrointestinal glomus tumor (GT1)

(A) Schematic representation of the MIR143-NOTCH2 fusion indicating the loci that are joint together; MIR143 exon 3 contains the miRNA precursor cluster, composed of pre-miR-143 and pre-miR-145 (the stem-loop structures, indicated with red and blue stars, respectively); (B) Experimental validation of the fusion by RT-PCR shows the junction sequence between exon 1 of MIR143 and exon 27 of NOTCH2; (C,D) FISH studies confirming break-apart signals in both NOTCH2 and MIR143 (Red, centromeric; Green, telomeric). (E) Long Range DNA PCR showing fusion of 13,278 bp of MIR143 intron 1 to the 444 bp of NOTCH2 intron 26; (F) Western blotting using NOTCH2 ICD antibody showing strong expression of a different size band (red arrow) in keeping with truncated NOTCH2 protein in GT1, compared to wild-type NICD protein seen in the control tumors angiosarcoma (AS) and gastrointestinal stromal tumor (GIST).

Fig. 2. MIR143-NOTCH1 gene fusion in a benign glomus tumor of the neck soft tissue (GT2)

(A) Typical morphologic appearance of a glomus tumor with uniform cuboidal cells with pale eosinophilic cytoplasm and round, bland nuclei, with a distinctive angiocentric growth around small blood vessels (H&E, 200x). (B) FISH analysis showing an unbalanced NOTCH1 rearrangement, with loss of the telomeric part (green signal) (tri-color assay, Orange/Green flanking NOTCH1, Red for C′-ABL used as control, centromeric to NOTCH1 at 9q34). (C) The top fusion candidate selected by FusionSeq was confirmed by RT-PCR showing the MIR143 exon 1 being fused to exon 27 of NOTCH1.

Fig. 3. MIR143-NOTCH3 fusion in a benign glomus tumor of the forearm (GT19)

(A) Histologic appearance of a benign glomus tumor; (B) Long-range DNA PCR showed the fusion of 11,844 bp of MIR143 intron 1 to 66 bp of NOTCH3 exon 29. (C,D) FISH analysis detected break-apart signals for MIR143 and NOTCH3, respectively (Red centromeric; Green, telomeric).

Fig. 4. Morphologic spectrum of pericytic tumors

(A) Index malignant gastrointestinal glomus tumor (GT1) showing transmural involvement; high power showing focal benign component with classic morphology (B), as well as areas of sarcomatous growth with areas of geographic necrosis (C); (D) Angioleiomyoma of the knee soft tissue area in a 32 year-old male, showing mature smooth muscle bundles proliferating out small vessel walls; this example was the only one of 18 examples tested showing a NOTCH2 gene rearrangement by FISH; (E) The index soft tissue myopericytoma (M1) showing multifocal presentation within subcutis by coronal STIR MRI and gross appearance (F); microscopically the tumor had a multinodular pattern, including intra-vascular growth (G) and high power showed ovale to short spindle cells in a haphazard, patternless pattern around small capillary vessels (H). (I) Digital glomus tumor in a patient with NF1 (GT18) showing dermal proliferation of perivascular cuboidal and bland oval cells (J), highlighted by SMA (K) and showing unbalanced rearrangement of NOTCH2 with deletion of telomeric part (Green signal) by FISH (L); (M) Malignant glomus tumor showing an abrupt transition from a benign monotonous appearance to a highly pleomorphic component (GT6) in the kidney; a different example in the stomach (GT7), showing focal areas of benign GT (N), while most of the peritoneal spread was composed of an undifferentiated spindle cell sarcoma morphology (O). The latter component showed low level of amplification of NOTCH2 centromeric parts (P, Red signal), with loss of the telomeric part (Green).

Fig. 5. MIR143-NOTCH2 fusion results in overexpression of 3′-NOTCH2 mRNA, triggered by the a strong MIR143 promoter, which is highly expressed in smooth muscle lineage

(A) Affymetrix U133A gene expression showing high levels of NOTCH2 mRNA expression in GT1 compared to M1 and other sarcoma types on X-axis (SS, synovial sarcoma; MLS, myxoid liposarcoma; MFH, malignant fibrous histiocytoma/undifferentiated pleomorphic sarcoma; LS, dedifferentiated liposarcoma; LMS, leiomyosarcoma; FS, adult type fibrosarcoma; CCS, clear cell sarcoma; AS, angiosarcoma); the Y-axis indicates the normalized expression of NOTCH2 mRNA (B) Real-Time PCR using 3′-NOTCH2 primers confirms the U133A high mRNA expression in GT1 compared to M1 and other tumors (GIST, LM, LMS, AS), while (C) Real-Time PCR with primers for the NOTCH2 ectodomain (outside the break) show low mRNA expression in GT1 compared to matched normal or other tumors. (D) Real-Time PCR for miR-143 expression show high levels in MIR143-fusion positive GT1 as well as in other smooth muscle neoplasms lacking MIR143 structural abnormalities; Y-axis for (B–D) represents the relative expression. (E) miRNA sequencing confirms the highly abundant expression of the miR143/miR145 genomic cluster across different smooth muscle tumors regardless of MIR143 rearrangement status (X-axis: GT#1,2,19, MIR143-fusion positive GTs; M1, myopericytoma; Myo1, infantile myofibromatosis; LM, leiomyoma; LMS, leiomyosarcoma; GIST, gastrointestinal stromal tumor; NF, normal fat; WDLS, well-differentiated liposarcoma; DDLS, dedifferentiated liposarcoma; Y-axis, relative frequency is obtained by the ratio of miRNA read counts by total miRNA reads per sample).