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. 2014:5:3116.
doi: 10.1038/ncomms4116.

Kinase fusions are frequent in Spitz tumours and spitzoid melanomas

Thomas Wiesner #  1   2 , Jie He #  3 , Roman Yelensky #  3 , Rosaura Esteve-Puig  4 , Thomas Botton  4 , Iwei Yeh  4 , Doron Lipson  3 , Geoff Otto  3 , Kristina Brennan  3 , Rajmohan Murali  1   5 , Maria Garrido  4 , Vincent A Miller  3 , Jeffrey S Ross  3 , Michael F Berger  1 , Alyssa Sparatta  4 , Gabriele Palmedo  6 , Lorenzo Cerroni  2 , Klaus J Busam  1 , Heinz Kutzner  6 , Maureen T Cronin  3 , Philip J Stephens  3 , Boris C Bastian  1   4   5
Affiliations

Kinase fusions are frequent in Spitz tumours and spitzoid melanomas

Thomas Wiesner et al. Nat Commun. 2014.

Abstract

Spitzoid neoplasms are a group of melanocytic tumours with distinctive histopathological features. They include benign tumours (Spitz naevi), malignant tumours (spitzoid melanomas) and tumours with borderline histopathological features and uncertain clinical outcome (atypical Spitz tumours). Their genetic underpinnings are poorly understood, and alterations in common melanoma-associated oncogenes are typically absent. Here we show that spitzoid neoplasms harbour kinase fusions of ROS1 (17%), NTRK1 (16%), ALK (10%), BRAF (5%) and RET (3%) in a mutually exclusive pattern. The chimeric proteins are constitutively active, stimulate oncogenic signalling pathways, are tumourigenic and are found in the entire biologic spectrum of spitzoid neoplasms, including 55% of Spitz naevi, 56% of atypical Spitz tumours and 39% of spitzoid melanomas. Kinase inhibitors suppress the oncogenic signalling of the fusion proteins in vitro. In summary, kinase fusions account for the majority of oncogenic aberrations in spitzoid neoplasms and may serve as therapeutic targets for metastatic spitzoid melanomas.

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Figures

Figure 1
Figure 1. Genomic aberrations identified in 38 spitzoid neoplasms by targeted sequencing.
(a) The columns denote the samples, the rows denote genes, purple squares represent gene fusions, red squares symbolize point mutations and indels, green squares denote gene amplifications, and purple squares indicate truncating mutations. The identified fusion genes and HRAS mutations were mutually exclusive in 38 spitzoid neoplasms. Mutations in PKHD1, ERBB4, LRP1B, and amplifications of MCL1 and CCNE1 are of unknown significance and co-occurred with kinase fusions and HRAS mutations. (b) Illustration of the distinct fusion genes for the ROS1, ALK, NTRK1, RET, and BRAF rearrangements. The grey bars represent the exons of the genes, the numbers below the bars the exon number, and the blue line the predicted breakpoints. The green shaded areas indicate the kinase domain and the blue shaded areas the coiled-coil domain of the fusion gene product. In the TP53-NTRK1 fusion transcript, multiple breakpoints spanning exon 8-12 of TP53 were predicted.
Figure 2
Figure 2. ROS1 fusions
(a) Histologic section of an atypical Spitz tumor with PWWP2A-ROS1 fusion from the gluteal region of a 55-year-old female (hematoxylin and eosin stain). Scale bar, 500μm. Scale bar magnification, 50μm. (b) Immunohistochemistry for ROS1 shows expression in the melanocytes; stromal cells serve as internal negative controls. Scale bar, 500μm. Scale bar magnification, 50μm. The FISH inset confirms the gene rearrangements using breakpoint flanking FISH probes. The rearranged ROS1 locus appears as individual green and red signals, and the wild type kinase allele with juxtaposed green/red signals. Scale bar, 10μm. (c) Illustration of the PWWP2A-ROS1 kinase fusion. ROS1 is located on chromosome 1q21, and PWWP2A on chromosome 5q33. Due to genomic rearrangements, exon 1 of PWWP2A is fused with exon 36 to 43 of ROS1, which contains the tyrosine kinase domain. The in-frame fusion junction of the transcript was confirmed by Sanger sequencing. (d) The PWWP2A-ROS1 fusion, but not the control-GFP construct, induces p-AKT, p-ERK, p-S6, and p-SHP2 in melan-a cells. Crizotinib inhibited at least partially the phosphorylation of the chimeric PWWP2A-ROS1 fusion protein, p-AKT, p-S6, and p-SHP2. The indicated protein weight markers in kDa are estimated from molecular weight standards. Results are representative of three independent experiments.
Figure 3
Figure 3. ALK fusions
(a) Histologic section of an atypical Spitz tumor excised from the upper arm of a 19-year-old male with a DCTN1-ALK fusion (hematoxylin and eosin stain). Scale bar, 500μm. Scale bar magnification, 50μm. (b) Immunohistochemistry shows ALK expression in the melanocytes; stromal cells serve as internal negative controls. Scale bar, 500μm. Scale bar magnification, 50μm. (c) FISH demonstrates the ALK gene rearrangement by the individual green and orange signals using breakpoint flanking probes. Scale bar, 10μm. (d) Illustration of the DCTN1-ALK kinase fusion. ALK is located on chromosome 1p23 and DCTN1 on chromosome 2p13. Due to genomic rearrangements, exon 1-26 of DCTN1 is fused with exon 20 to 29 of ALK, which contains the tyrosine kinase domain. The in-frame junction of the fusion transcript was confirmed with Sanger sequencing. (e) The DCTN1-ALK fusion construct, but not the control-GFP construct, induces p-AKT, p-ERK and p-S6 in melan-a cells. These effects and the phosphorylation of chimeric DCTN1-ALK protein can be inhibited with crizotinib. The indicated protein weight markers in kDa are estimated from molecular weight standards. Results are representative of three independent experiments.
Figure 4
Figure 4. NTRK1 fusions
(a) Histologic section of a spitzoid melanoma excised from the left knee of a 39-year-old woman with an LMNA-NTRK1 fusion (hematoxylin and eosin stain). Scale bar, 500μm. Scale bar magnification, 50μm. (b) Immunohistochemistry demonstrates the NTRK1 expression in melanocytes; stromal cells serve as internal negative controls. Scale bar, 500μm. Scale bar magnification, 50μm. The FISH inset confirms the gene rearrangements using breakpoint flanking FISH probes by the individual green and red signals. Scale bar, 10μm. (c) The LMNA-NTRK1 kinase fusion is caused by a 743kb deletion on chromosome 1q, joining the first 2 exons of LMNA with exon 11 to 17 of NTRK1. The in-frame junction of the fusion transcript was confirmed with Sanger sequencing. (d) The LMNA-NTRK1 fusion construct, but not the full-length, wild-type NTRK1 or the control-GFP constructs induce p-AKT, p-ERK, pS6 and p-PLCγ1 in melan-a cells. A small molecule kinase inhibitor, AZ-23, inhibited the phosphorylation of LMNA-NTRK1 and the activation of the oncogenic signaling pathways. The indicated protein weight markers in kDa are estimated from molecular weight standards. Results are representative of three independent experiments.
Figure 5
Figure 5. RET fusions
(a) Histologic section of a pigmented spindle cell nevus (a morphologic variant of Spitz nevus) from a 50-year-old woman with a GOLGA5-RET fusion (hematoxylin and eosin stain). Scale bar, 500μm. Scale bar magnification, 50μm. (b) RET expression in melanocytes; keratinocytes serve as internal negative controls. Scale bar, 100μm. The individual green and red signals in FISH confirm the gene rearrangements using breakpoint flanking FISH probes. Scale bar, 10μm. (c, d) The GOLGA5-RET construct, but not the wild-type, full-length RET or the control-GFP constructs, induces p-AKT, p-ERK, p-S6, and p-PLCγ1 in melan-a cells. The activation of these pathways and the phosphorylation of GOLGA5-RET can be inhibited with (c) vandetanib and (d) cabozantinib. The indicated protein weight markers in kDa are estimated from molecular weight standards. Results are representative of three independent experiments.
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