Activation of the GLI oncogene through fusion with the beta-actin gene (ACTB) in a group of distinctive pericytic neoplasms: pericytoma with t(7;12)

Abstract

Activation of the GLI oncogene is an important step in the sonic hedgehog signaling pathway, and leads to, eg, tissue-specific cell proliferation during embryogenesis. GLI activity in adult tissues is restricted, but has been identified in various neoplasms, as a result of mutations in the PTCH (patched) or SMOH (smoothened) genes, encoding components of the sonic hedgehog pathway, or by amplification of GLI. Herein, we present a new mechanism of GLI activation through fusion with the beta-actin gene (ACTB) in five histologically distinctive soft tissue tumors showing a t(7;12)(p21-22;q13-15) and a pericytic phenotype. Each was composed of a perivascular proliferation of monomorphic short spindle cells that stained positively for smooth muscle actin and laminin and that showed pericytic features by electron microscopy. To date, with a median follow-up of 24 months, none has behaved in an aggressive manner. Molecular genetic analysis showed that the translocation in all cases resulted in a fusion transcript including the 5'-part of ACTB and the 3'-part of GLI. The DNA-binding zinc finger domains of GLI were retained in the fusion transcripts and it is likely that the replacement of the promoter region of GLI with that of the ubiquitously expressed ACTB gene leads to deregulation of GLI expression and its downstream target genes.

Figure 1

a: Position of the selected primers in relation to the ACTB and GLI cDNA sequences (see text and Table 1 for details). The exons as well as primer suffix and orientation (F, forward; R, reverse) are indicated. b: Schematic representation of the ACTB-GLI and GLI-ACTB fusion transcripts, detected by RT-PCR in five cases with a t(7;12)(p21-22;q13-15). Exons are illustrated as boxes, intronic material as thick horizontal lines. Exons 7 to 10 of GLI, encoding DNA-binding zink finger domains, are highlighted.

Figure 2

Tumors were composed of palely eosinophilic spindle-to-ovoid cells with a lobular/infiltrative margin (A). Tumor cells in each case were monomorphic and arranged around numerous thin-walled capillary vessels (B), which were better highlighted by immunostaining for CD34 (C). Subendothelial proliferation of tumor cells, as often seen in myopericytic tumors and mimicking vascular invasion, was seen in two cases (D). Tumor cells showed consistent, albeit variably prominent immunopositivity for smooth muscle actin (E) and there was pericellular positivity for laminin (F). Capillary vessel walls are also highlighted by laminin staining.

Figure 3

Electron microscopic examination closely apposed lesional cells arranged around small vessels (V). Tumor cells had prominent external lamina and subplasmalemmal densities (black arrows). Also noted were bundles of cytoplasmic myofilaments (black arrowheads) with focal densities, as well as intermediate-type intercellular junctions (white arrowheads). These appearances, in context, favor pericytic differentiation.

Figure 4

a: FISH images of cases 1 and 5, showing split signals (arrows) with the 7p22 (RP11-1275H24 and RP11-93G19)- and 12q13 (RP11-772E1 and RP11-181L23)-specific probes in cells with the t(7;12)(p22;q13), strongly suggesting that the translocation breakpoints were located within the segments covered by these BACs. The partial karyotype illustrates the t(7;12)(p22;q13) as seen in case 1. b: Physical map of 7p22, including RP11-1275H24 and RP11-93G19, flanking BAC probes and the genes ACTB and FLJ11467. The exons (filled boxes) and the 5′→ 3′ orientation of the genes are indicated. BAC probe RP11-1275H24 is, based on our FISH mapping and PCR showing that the ACTB is present, substantially larger than the ∼85 kb reported at the NCBI Nucleotide Browser (http//:www.ncbi.nlm.nih.gov). c: Physical map of 12q13, including RP11-181L23 and RP11-772E1, flanking BACs, and the genes DDIT3 and GLI1.

Figure 5

RT-PCR detecting ACTB-GLI fusion transcripts in cases 1 to 5 (case 1: ACT61F-GLI868R, 270 bp; case 2: ACT18F-GLI1246R, 754 bp; case 3: ACT61F-GLI1477R, 1172 bp; case 4: ACT61F-GLI938R, 484 bp; case 5: ACT106F-GLI868R, 610 and 712 bp), and GLI-ACTB fusion transcripts in cases 1and 5 (case 1: GLI520F-GLI520R, 289 bp; case 5: GLI412F-ACT520R, 162 bp). M, 100-bp ladder; T, tumor.

Figure 6

Partial nucleotide sequences spanning the breakpoints in the ACTB-GLI and GLI-ACTB fusion transcripts. ACTB sequences are highlighted in bold characters, and amino acids are written in capital letters. In case 1, the inserted guanine that maintains the open reading frame in the ACTB-GLI fusion is indicated (arrow). In case 5, the out-of-frame GLI-ACTB fusion leads to an S→R substitution (arrow) in the GLI sequence and ultimately to the introduction of a premature stop codon (asterisk). See text for details.