TPM3-ALK and TPM4-ALK oncogenes in inflammatory myofibroblastic tumors

Abstract

Inflammatory myofibroblastic tumors (IMTs) are neoplastic mesenchymal proliferations featuring an inflammatory infiltrate composed primarily of lymphocytes and plasma cells. The myofibroblastic cells in some IMTs contain chromosomal rearrangements involving the ALK receptor tyrosine-kinase locus region (chromosome band 2p23). ALK-which is normally restricted in its expression to neural tissues-is expressed strikingly in the IMT cells with 2p23 rearrangements. We now report a recurrent oncogenic mechanism, in IMTs, in which tropomyosin (TPM) N-terminal coiled-coil domains are fused to the ALK C-terminal kinase domain. We have cloned two ALK fusion genes, TPM4-ALK and TPM3-ALK, which encode approximately 95-kd fusion oncoproteins characterized by constitutive kinase activity and tyrosylphosphorylation. Immunohistochemical and molecular correlations, in other IMTs, implicate non-TPM ALK oncoproteins that are predominantly cytoplasmic or pre- dominantly nuclear, presumably depending on the subcellular localization of the ALK fusion partner. Notably, a TPM3-ALK oncogene was reported recently in anaplastic lymphoma, and TPM3-ALK is thereby the first known fusion oncogene that transforms, in vivo, both mesenchymal and lymphoid human cell lineages.

Figure 1.

ALK subcellular localization (A) and FISH evaluations (B–C) in IMT case 1: Strong cytoplasmic ALK staining is evident in the spindle cells, whereas ALK is undetectable in the intervening inflammatory cells (A). FISH probes centromeric and telomeric in relationship to ALK are detected with Spectrum Green and Spectrum Orange, respectively (B-C). B: Centromeric and telomeric ALK probes are translocated apart in the top two cells. Bottom two cells, presumably inflammatory, have normal FISH signals. C: A chromosome band 19p13 yeast artificial chromosome clone, 766_E_7, centromeric to the TPM4 gene, is detected with 50:50 Texas Red: fluorescein isothiocyanate. Small arrows indicate colocalized yeast artificial chromosome 766_E_7 (yellow) and telomeric/red ALK probe, whereas large arrows indicate corresponding centromeric/green ALK probe. The two cells at right have normal FISH signals.

Figure 2.

Expression of alternate TPM4-ALK transcripts in IMT case 1. Type 1 TPM4-ALK was identified by RACE, whereas the type 2 transcript was predicted based on published TPM4 sequence. Both type 1 (609 bp) and type 2 (716 bp) transcripts were identified by RT-PCR (A). Primer locations are as schematized (B).

Figure 3.

A: Expression of TPM4-ALK and TPM3-ALK in 11 IMT. RT-PCR with universal TPM4/TPM3 primers identifies 147-bp transcripts in IMT cases 1, 2, and 3 (top), whereas positive control (177 bp) GAPDH transcripts are amplified from all cases (bottom). B: RT-PCR with primers specific for TPM3-ALK reveals fusion transcripts (130 bp) in cases 2 and 3. The identity of all transcripts was confirmed by sequencing (data not shown).

Figure 4.

Homology between TPM4-ALK and TPM3-ALK cDNA fusion regions. Sequence mismatches, used to distinguish the TPM4-ALK and TPM3-ALK RT-PCR products, are set in boxes. ALK sequence is shown in a gray box.

Figure 5.

In vitro kinase (A), phosphotyrosine (B), and ALK (C) evaluations in IMT case 1 (TPM4-ALK), IMT case 2 (TPM3-ALK), and a non-IMT spindle-cell sarcoma. Three kinase-active, tyrosylphosphorylated ALK forms (80 to 95 kd) are seen in the IMT. Native ALK (200 kd), seen in IMT case 1 and also demonstrable in case 2 at longer exposures, lacked constitutive kinase activity and tyrosylphosphorylation.