ALK oncoproteins in atypical inflammatory myofibroblastic tumours: novel RRBP1-ALK fusions in epithelioid inflammatory myofibroblastic sarcoma

Abstract

ALK oncogenic activation mechanisms were characterized in four conventional spindle-cell inflammatory myofibroblastic tumours (IMT) and five atypical IMT, each of which had ALK genomic perturbations. Constitutively activated ALK oncoproteins were purified by ALK immunoprecipitation and electrophoresis, and were characterized by mass spectrometry. The four conventional IMT had TPM3/4-ALK fusions (two cases) or DCTN1-ALK fusions (two cases), whereas two atypical spindle-cell IMT had TFG-ALK and TPM3-ALK fusion in one case each, and three epithelioid inflammatory myofibroblastic sarcomas had RANBP2-ALK fusions in two cases, and a novel RRBP1-ALK fusion in one case. The epithelioid inflammatory myofibroblastic sarcoma with RRBP1-ALK fusion had cytoplasmic ALK expression with perinuclear accentuation, different from the nuclear membranous ALK localization in epithelioid inflammatory myofibroblastic sarcomas with RANBP2-ALK fusions. Evaluation of three additional uncharacterized epithelioid inflammatory myofibroblastic sarcomas with ALK cytoplasmic/perinuclear- accentuation expression demonstrated RRBP1-ALK fusion in two cases. These studies show that atypical spindle-cell IMT can utilize the same ALK fusion mechanisms described previously in conventional IMT, whereas in clinically aggressive epithelioid inflammatory myofibroblastic sarcoma we identify a novel recurrent ALK oncogenic mechanism, resulting from fusion with the RRBP1 gene. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Keywords: ALK; RRBP1; epithelioid inflammatory myofibroblastic sarcoma; fusion gene; inflammatory myofibroblastic tumour; mass spectrometry.

Figure 1

ALK immunoprecipitations were immunoblotted for phosphotyrosine (top) and ALK (middle) and were electrophoresed and stained with Coomassie blue (bottom). Coomassie blue bands indicated by arrows corresponded in size to the putative ALK fusion proteins and were excised and subjected to mass spectrometry analyses.

Figure 2

A: Each of four aberrant ALK proteins characterized by mass spectrometry was a fusion protein in which the ALK fusion partner juxtaposed coiled coil oligomerization domains to the ALK tyrosine kinase domain. Peptides mapping to ALK and the fusion partners are indicated by red and green bars, respectively. B: The fusions were confirmed by RT-PCR and Sanger sequencing. Chromosomal cytoband locations of the ALK-fusion partner genes are as indicated in parentheses (for TPM3: 1q21.3). The RRBP1-ALK fusion incorporates an alternate splicing 33-bp intronic sequence fused to intra-exonic sequence from ALK exon 20, maintaining the open reading frame.

Figure 3

Schematic of RRBP1-ALK domains. The dashed line indicates the breakpoints. This model is predicted using the Simple Modular Architecture Research Tool (SMART; http://smart.embl-heidelberg.de/) and the Human Protein Reference Database (http://www.hprd.org/). The ALK transmembrane domain (TM) is a cell membrane transmembrane whereas the RRBP1 TM is an organelle (most likely endoplasmic reticulum) domain.

Figure 4

Cases A8 and A9 show histological features of EIMS, with mixed lymphocyte, neutrophil, and eosinophil infiltration (A), while expressing cytoplasmic ALK protein with perinuclear accentuation by immunohistochemistry (B). FISH demonstrated RRBP1-ALK fusion in both cases (C: A8 & D: A9).