Kinase fusions are frequent in Spitz tumors and spitzoid melanomas

Abstract

Spitzoid neoplasms are a group of melanocytic tumors with distinctive histopathologic features. They include benign tumors (Spitz nevi), malignant tumors (spitzoid melanomas), and tumors with borderline histopathologic features and uncertain clinical outcome (atypical Spitz tumors). Their genetic underpinnings are poorly understood, and alterations in common melanoma-associated oncogenes are typically absent. Here we show that spitzoid neoplasms harbor 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 signaling pathways, are tumorigenic, and are found in the entire biologic spectrum of spitzoid neoplasms, including 55% of Spitz nevi, 56% of atypical Spitz tumors, and 39% of spitzoid melanomas. Kinase inhibitors suppress the oncogenic signaling 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.

Melanocytic neoplasms comprise several tumor types that are characterized by distinct clinical, pathologic, and genetic features. The clinical course of melanocytic tumors may be indolent (benign nevi), aggressive (malignant melanomas), or intermediate (melanocytic tumors of uncertain malignant potential).

In 1948, Sophie Spitz coined the term ‘melanoma of childhood’ for a group of melanocytic skin tumors composed of spindled or epithelioid melanocytes that developed predominantly in children and adolescents.¹ It later became clear that these tumors could also arise later in life, and that the majority of these neoplasms behaved in an indolent fashion, which led to the introduction of the term ‘Spitz nevus’ to indicate their benign nature. Malignant tumors with spitzoid histologic features were termed ‘spitzoid melanomas’, and these tumors often showed aggressive clinical behavior with widespread metastasis, similar to conventional melanomas. Tumors with histologic features overlapping those of Spitz nevi and spitzoid melanoma have been termed ‘atypical Spitz tumors’. Atypical Spitz tumors have the capacity to metastasize, but this is usually limited to the regional lymph nodes, and has little effect on patient survival.^2,3

Genetic alterations in the majority of spitzoid neoplasms are not known, and they lack mutations in melanoma-associated oncogenes such as NRAS, KIT, GNAQ or GNA11.⁴ However, subsets of spitzoid neoplasms characterized by distinct histopathologic features show HRAS mutations⁵, or BRAF mutations combined with bi-allelic BAP1 loss^6,7, suggesting that activation of kinase pathways plays an important role in the pathogenesis of these tumors. Identification of additional genetic events in kinase pathways may contribute to a better understanding of the pathogenesis of spitzoid neoplasms, and may facilitate the development of effective targeted therapies for metastasizing tumors, as illustrated by the success of small molecule kinase inhibitors in prolonging the lives of patients with a broad range of malignancies.^8-10

For diagnostic reasons, spitzoid neoplasms are usually formalin-fixed and paraffin-embedded (FFPE) in their entirety, and nucleic acids isolated from FFPE tissue are typically degraded and of suboptimal quality. In addition, the percentage of neoplastic cells in spitzoid neoplasms is frequently low, due to the presence of varying numbers of admixed non-neoplastic lymphocytes, fibroblasts, and keratinocytes. The suboptimal quality of extracted nucleic acids and the low proportion of neoplastic melanocytic cells makes the identification of genetic aberrations in spitzoid neoplasms challenging and therefore a high sequencing coverage is necessary. For these reasons, we chose a massively parallel sequencing approach with high sequencing coverage for previously described cancer genes to investigate genetic aberrations in spitzoid neoplasms.

Our genomic analysis of 140 spitzoid neoplasms reveals gene rearrangements of the kinases ROS1 (17%; n=24), NTRK1 (16%; n=23), ALK (10%; n=14), BRAF (5%; n=7), and RET (3%; n=4) resulting in in-frame kinase fusions. These kinase fusions occur across the entire biologic spectrum of spitzoid neoplasms, including Spitz nevi (55%, fusions in 41 of 75 cases), atypical Spitz tumors (56%; fusions in 18 of 32 cases), and spitzoid melanomas (39%; fusions in 13 of 33 cases). The chimeric proteins appear in a mutually exclusive pattern, are constitutively active, stimulate oncogenic signaling pathways, are tumorigenic and may serve as diagnostic markers and as therapeutic targets for aggressive or metastasizing Spitz tumors.

RESULTS

Identification of Kinase Fusions

We investigated genomic alterations in a discovery cohort of 30 Spitz nevi and 8 atypical Spitz tumors by targeted massively parallel sequencing (Supplementary Table S1 summarizes the clinical, histopathologic and genetic data). For targeted DNA sequencing, we sequenced 3230 exons from 182 cancer-related genes and 37 introns of 14 genes commonly rearranged genes in cancer. We obtained an average unique coverage of 997x, with 99.96% of exons being sequenced at ≥100x coverage. In addition, we performed targeted transcriptome sequencing for 612 transcripts of kinases and kinase-related genes and achieved an average of 56,588,548 unique read pairs per spitzoid neoplasm. Six tumors (16%) harbored HRAS c.182A>T (p.Q61L) mutations and exhibited histopathologic features characteristic of the previously-described HRAS-mutant Spitz nevus variant.⁵ Additionally, we found mutations in PKHD1, ERBB4, LRP1B, and amplifications of MCL1 and CCNE1 in one case each (Fig. 1a). No sequence alterations were found in the known melanoma oncogenes BRAF, NRAS, KIT, GNAQ, or GNA11 or in other cancer-related genes listed in Supplementary Table 3.

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.

Kinase fusions were identified in 18 of 30 (60%) Spitz nevi and in 6 of 8 (75%) atypical Spitz tumors (Fig. 1a and 1b). The rearrangements included gene fusions of the membrane-bound receptor tyrosine kinases ROS1 (n=11, 29%), ALK (n=6, 16%), NTRK1 (n=3, 8%), and RET (n=2, 5%). Two spitzoid neoplasms harbored t(7;19)(q34;q13) translocations that involved the serine/threonine kinase BRAF, but with different 5’-partners (CEP89 and LSM14A) in each tumor. Using RT-PCR with breakpoint flanking primers, we verified that the fusion genes were expressed and produced in-frame transcripts. Using interphase FISH with breakpoint-flanking probes, we confirmed the disruptions at the ROS1, ALK, NTRK1, RET, and BRAF loci.

We validated our findings in an independent cohort of 102 spitzoid neoplasms using interphase FISH (Supplementary Table S2 summarizes the clinical, histopathologic and genetic data). In total, we analyzed 140 spitzoid neoplasms and identified fusions in 41 of 75 (55%) Spitz nevi, in 18 of 32 (56%) atypical Spitz tumors, and in 13 of 33 (39%) spitzoid melanomas (Table 1). We confirmed the expression of the chimeric ROS1, NTRK1, ALK, and RET proteins using immunohistochemistry. Cases harboring translocations showed moderate to strong staining for the corresponding fusion kinase, which was not observed in the cases lacking gene rearrangements. Patients with translocation-positive spitzoid neoplasms were younger (median 21 years) than patients whose tumors did not harbor translocations (median 32.5 years; Mann Whitney test P<0.001).

Table 1.

Frequency of kinase fusions in spitzoid neoplasms.

Fusion	Spitz nevus (n=75) % (number of cases)	Atypical Spitz tumor (n=32) % (number of cases)	Spitzoid melanoma (n=33) % (number of cases)	Total (n=140) % (number of cases)
ROS1	25.3% (19)	6.3% (2)	9.1% (3)	17.1% (24)
ALK	10.7% (8)	15.6% (5)	3% (1)	10% (14)
NTRK1	10.7% (8)	25% (8)	21.2% (7)	16.4% (23)
BRAF	5.3% (4)	6.3% (2)	3% (1)	5% (7)
RET	2.7% (2)	3.1% (1)	3% (1)	2.9% (4)
Total	54.7% (41)	56.3% (18)	39.4% (13)	51.4% (72)

ROS1 Fusions

ROS1 rearrangements were found in 19 of 73 (26%) Spitz nevi, 3 of 34 (8%) atypical Spitz tumors, and 3 of 33 (9%) spitzoid melanoma (Fig. 2a). The ROS1 rearrangements fused the intact tyrosine kinase coding sequence of ROS1 to the 5’ portion of nine different partners (Fig. 1a and Supplementary Fig. S1-9). The expression of the chimeric ROS1 protein by immunohistochemistry was observed exclusively in cases with ROS1 rearrangements (Fig. 2b and 2c). Expression of the PWWP2A-ROS1 fusion construct in melan-a cells showed increased phosphorylation of the fusion protein, suggesting that the chimeric protein is constitutively active. Consistent with this, the MAPK and PI3K pathways were strongly activated compared to the GFP and the wild-type, full-length ROS1 controls. The phosphorylation of PWWP2A-ROS1, AKT, S6 and SHP2, but not ERK, was at least partially inhibited by crizotinib, an FDA-approved drug for lung cancer with ALK translocations that acts as an ALK⁹ and ROS1¹² inhibitor (Fig. 2d).

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.

ALK Fusions

We found ALK fusions in 8 of 75 (10.7%) Spitz nevi, 5 of 32 (15.6%) atypical Spitz tumors, and 1 of 33 (3%) spitzoid melanoma (Fig. 3a). Expression of the chimeric ALK protein was confirmed by immunohistochemistry and the rearrangements were validated in all cases with ALK fusions with FISH (Fig. 3b and 3c). Cases without ALK rearrangements by sequencing did not show ALK expression in immunohistochemistry or separated signals by FISH. TPM3 and DCTN1 were 5’ fusion partners for ALK rearrangements (Fig. 1b and Supplementary Fig. S10-11). TPM3-ALK fusions were detected in 8 spitzoid neoplasms. DCTN1 represents a novel fusion partner of ALK, was found in 6 cases, and resulted from a balanced translocation between homologous copies of chromosome 2 (Fig. 3d). Expression of the DCTN1-ALK fusion construct in melan-a cells showed increased phosphorylation of the fusion protein compared to the wild-type, full-length ALK, suggesting that the chimeric protein is constitutively active. Similar to the ROS1 fusions, AKT, ERK, and S6 were more phosphorylated in melan-a cells with the ALK fusion construct compared to the GFP control. Crizotinib inhibited the ALK fusion-induced activation of the oncogenic PI3K and MAPK signaling pathways (Fig. 3e).

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.

NTRK1 Fusions

NTRK1 rearrangements were detected in 8 of 75 (10.7%) Spitz nevi, 8 of 32 (25%) atypical Spitz tumors, and 7 of 33 (21.2%) spitzoid melanoma (Fig. 4a). The expression of NTRK1 was confirmed with immunohistochemistry in cases with NTRK1 fusions (Fig. 4b), but was absent in cases without NTRK1 rearrangements. The LMNANTRK1 fusions involved the novel fusion partner LMNA (localized at 1q22), and were caused by a 743 kb deletion of chromosome 1q, joining the first two exons of LMNA with NTRK1 starting at exon 11 (Fig. 4c). The interchromosomal TP53-NTRK1 translocation resulted from a fusion joining NTRK1 starting from exon 9, containing the tyrosine kinase domain, to the 3'UTR of TP53 (Fig. 1B and Supplementary Fig. S12-13). The expression of the LMNA-NTRK1 fusion construct in melan-a cells showed increased levels of phosphorylation of the fusion protein, AKT, ERK, S6 and PLCγ1 compared to the control cells. The strong activation of MAPK, PLCγ1 and PI3K pathways was inhibited by the NTRK1 inhibitor AZ-23 (Fig. 4d).

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.

RET Fusions

We found RET fusions in 2 of 75 (2.7%) Spitz nevi, 1 of 32 (3.1%) atypical Spitz tumors, and 1 of 33 (3%) spitzoid melanoma (Fig. 5a). Fusions of RET on chromosome 10q11 involved the 5’ fusion partners KIF5B on chromosome 10p11 and GOLGA5 on chromosome 14q32 (Fig. 1b and Supplementary Fig. S14-15). In both fusions, the RET tyrosine kinase domain was fused to the coiled-coil domains of KIF5B (exons 1–16) or GOLGA5 (exons 1-7). Immunohistochemical expression of RET was observed only in cases with RET translocations (Fig. 5b). Expression of the GOLGA5-RET fusion construct in melan-a cells showed increased phosphorylation of the fusion protein, AKT, ERK, S6, and PLCγ-1 compared to control cells. The phosphorylation of these proteins could be suppressed by vandetanib (Fig. 5c) or cabozantinib (Fig. 5d), which are both small molecule RET inhibitors that are FDA approved for medullary thyroid cancer.

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.

BRAF Fusions

We identified gene rearrangements of the serine/threonine kinase BRAF in 4 of 75 (5.3%) Spitz nevi, 2 of 32 (6.3%) atypical Spitz tumors, and 1 of 33 (3%) spitzoid melanoma. The fusion genes contained CEP89 exons 1–16 followed by the kinase domain of BRAF encoded by exons 9–18, or LSM14A exons 1-9 followed by BRAF exons 9-18, both resulting in loss of the auto-inhibitory, N-terminal RAS-binding domain of BRAF (Fig. 1b and Supplementary Fig. S16-17). In addition to the identified translocations, 1 of 75 (1.3%) Spitz nevi and 1 of 32 (3.1%) atypical Spitz tumors showed BRAF amplification as determined by at least 8 BRAF fusion signals per nucleus in interphase FISH.

Functional validation in xenograft models

To validate the oncogenic roles of the novel fusion kinases, we stably transduced mouse melan-a cells with PWWP2A-ROS1, DCTN1-ALK, LMNA-NTRK1, GOLGA5-RET fusions. We then injected the transduced cells subcutaneously and bilaterally into the flank of immunocompromised mice. We monitored the mice for the tumor formation every 3 to 4 days. Within 40 days, all injection sites in the fusion kinase groups (PWWP2A-ROS1, DCTN1-ALK, LMNA-NTRK1, GOLGA5-RET) developed rapidly growing tumors similarly to the one observed at the injection sites of melanocytes expressing the known melanoma oncogenes HRAS^G12V and NRAS^G12V (Supplementary Fig. S18). We observed no tumors at the injection sites in the control group with melanocytes that had been transduced with GFP.

DISCUSSION

Our results show that kinase fusions are important mechanisms of oncogene activation in spitzoid neoplasms. In aggregate, 72 of 140 (51.4%) spitzoid neoplasms harbored fusions involving the receptor tyrosine kinases ROS1 (17%), NTRK1 (16%), ALK (10%), RET (3%), and the serine/threonine kinase BRAF (5%). All fusions occurred in a mutually exclusive pattern (Fig. 1a), and no fusions were detected in tumors with HRAS mutations. All of the sequenced kinase fusions created chimeric proteins that retained the intact kinase domain at the 3’ end of the fusion transcript (Fig. 1b), a pattern similar to that observed in other cancers that harbor rearrangements involving these kinases.^13-15 All recombination sites between kinases and translocation partners involved the canonical intronic recombination sites, similar to those described in other types of cancers (ROS1: intron 33-35; ALK: intron 19; NTRK1: intron 8-10; RET: intron 11; BRAF: intron 8).¹⁶ The majority of the novel 5’ fusion partners identified in our study, namely those of ROS1 (PWWP2A, PPFIBP1, ERC1, MYO5A, CLIP1, HLA-A, KIAA1598 and ZCCHC8), ALK (DCTN1), NTRK1 (LMNA), and BRAF (CEP89 and LSM14A) contributed coiled-coil domains to the fusion proteins. The coiled-coil domains may play similar roles to the established 5’ fusion partners (TPM3¹⁷, GOLGA5¹⁸, KIF1B¹³) of these kinases in promoting dimerization¹⁹ and auto-activation of the kinases.²⁰

Rearrangements of the kinases ROS1, ALK, NTRK1, RET and BRAF with similar fusion sites have previously been described in various types of aggressive tumors. ROS1 fusions have been described in various cancer types, including lung carcinoma²¹, glioblastoma²², and cholangiocarcinoma.²³ ALK fusions have been described in anaplastic large cell lymphoma¹⁷, lung cancer¹⁴, inflammatory myofibroblastic tumors²⁴ and anecdotal studies have also reported ALK fusions in acral melanoma.²⁵ RET fusions, including the pericentric inversions giving rise to the KIF5B-RET fusion observed in spitzoid neoplasms, have been shown to drive lung cancer formation^13,15,26 and an interchromosomal GOLGA5-RET rearrangement was first described in papillary thyroid carcinomas occuring in children exposed to radioactive fallout from the Chernobyl nuclear accident.¹⁸ BRAF fusions have been identified in pilocytic astrocytomas²⁷, papillary thyroid carcinoma²⁸, and rarely in melanocytic tumors.^29,30 In addition, our data that patients with translocation-positive spitzoid neoplasms were younger than patients whose tumors lacked translocations is similar to observations that lung cancers¹⁵, thyroid cancers¹⁸, and astrocytomas²⁷ harboring kinase fusions are more common in younger patients.

Our findings of the activated effector pathways downstream of the fusion kinases are in line with other experimental systems of cancer. For example, ubiquitous expression of constitutively active RFP-RET fusions in mice results in generalized melanocyte proliferation, formation of nevi and ultimately melanomas with MAPK pathway activation; this is similar to our finding of GOLGA5-RET fusions in spitzoid neoplasms.³¹ Autocrine neurotrophin signaling involving NTRK1 has been shown to promote proliferation and migration in melanoma cell lines³², and constitutively active NTRK1 activates MAPK and PI3K pathways in NIH-3T3 cells.³³ Phosphorylation of the MAPK and PI3K pathways is an essential component of oncogenic ALK signaling in lymphoma¹⁷ and lung cancer¹⁴, and we found that the novel fusion DCTN1-ALK has similar effects. The activation of the MAPK and PI3K pathways by the novel PWWP2A-ROS1 fusion is also similar to the changes associated with other oncogenic ROS1 fusions in various cancer types.^12,34 The results of our functional validation experiments in xenograft models are consistent with previous studies which showed that TPM3-ROS1¹⁵, EML4-ALK¹⁴, KIF5B-RET^13,15,26 fusions drive tumor formation in non-small cell lung cancers.

The common occurrence of kinase fusions in the entire biologic spectrum of spitzoid neoplasms (benign Spitz nevi, atypical Spitz tumors, spitzoid melanomas) suggests that these fusions occur early in the pathogenesis of spitzoid neoplasms. Therefore, kinase fusions are necessary, but not sufficient for malignant transformation, a situation analogous to that of mutations in oncogenes (such as BRAF, NRAS, GNAQ and GNA11) commonly found in melanocytic neoplasms. Consequently, the frequent kinase fusions described herein are unlikely to be useful in distinguishing benign from malignant spitzoid neoplasms.

Although the majority of spitzoid neoplasms behave in an indolent fashion, some spitzoid neoplasms metastasize and require systemic therapy. Lung cancers, lymphomas, and sarcomas with ALK or ROS1 fusions, and thyroid cancers with RET fusions can be successfully treated using US Food and Drug Administration (FDA)-approved kinase inhibitors such as crizotinib, cabozantinib, and vandetanib. The fusion kinases identified here are therefore potential therapeutic targets for translocation-positive melanocytic tumors that require systemic therapy.

METHODS

Patients

The study was conducted according to the Declaration of Helsinki and human tissues were obtained with patient-informed consent under approval by the Institutional Review Boards/Ethics Committees of Memorial Sloan-Kettering Cancer Center, New York, the University of California, San Francisco, and the Medical University of Graz, Austria. The excised skin lesions were fixed in 4% neutral buffered formalin, embedded in paraffin, processed using routine histologic methods and stained with hematoxylineosin. Specimens were collected over a time period of 6 months and the histopathologic diagnosis of Spitz nevus, atypical Spitz tumor, and spitzoid melanoma was confirmed by at least three dermatopathologists. Specimens with insufficient tissue amount or severely degraded nucleic acids were excluded. In total, specimens from 140 patients were analyzed and the clinical, histologic, and genetic characteristics are summarized in Supplementary Tables S1 and S2.

Targeted DNA sequencing

We sequenced 3230 exons from 182 cancer-related genes (Supplementary Table S3) and 37 introns of 14 genes (Supplementary Table S4) commonly rearranged genes in cancer with average depth-of-coverage of greater than 500x. Prior to DNA extraction, FFPE samples from all cases were reviewed to confirm that the tissue was of sufficient size to generate a minimum of 50ng of DNA and that this DNA would be derived from areas that contained a minimum of 20% melanocytic nuclei. DNA was isolated from 40μm thick sections of formalin-fixed, paraffin-embedded (FFPE) tissue. DNA sequencing was performed on indexed, adaptor ligation, hybridization-captured libraries (Agilent SureSelect custom kit). Sequencing was performed on the HiSeq-2000 instrument (Illumina), with 49x49 paired reads to an average depth of 997x (Supplementary Table S5).

RNA sequencing

Total RNA extracted from 40μm thick sections of FFPE tumor was reverse-transcribed with random hexamer primers using the SuperScript® III First-Strand Synthesis System (Invitrogen). Double stranded cDNA was synthesized with the NEBNext® mRNA Second Strand Synthesis Module (New England Biolabs).³⁵ Hybrid selection of indexed, adaptor-ligated libraries was performed using the cDNA Kinome hybridization kit with 612 transcripts of kinases and kinase-related genes (Agilent SureSelect Human Kinome Kit).³⁶ Selected libraries were sequenced on the HiSeq-2000 instrument (Illumina) with 49 x 49 paired reads. For RNA sequencing, we used a sequencing approach targeting 612 transcripts of kinases and kinase-related genes. We aimed for a high number of unique read pairs (≥ 50,000,000) per sample (Supplementary Table S6).

Sequence data analysis

Sequence data from gDNA and cDNA was mapped to the reference human genome (hg19) using the BWA aligner and processed using publically available SAMtools³⁷, Picard (http://picard.sourceforge.net) and GATK³⁸. Genomic base substitutions and indels were detected using custom tools optimized for mutation calling in heterogeneous tumor samples, based on detecting common sequence variations and local sequence assembly. Variations were filtered using dbSNP and a custom artifact database, then annotated for known and likely somatic mutations using COSMIC. Copy number alterations were detected by comparing targeted genomic DNA sequence coverage with a process-matched normal control sample. Genomic rearrangements were detected by clustering chimeric reads mapping to targeted introns/exons. Expression levels were determined by analyzing cDNA sequence coverage of targeted exons.

Gene fusion confirmation

All gene fusions were validated by qRT-PCR from cDNA using the primers listed in Supplementary Table S7 and analysis on a Bioanalyzer (Agilent). Specific PCR amplicons were only detected with the appropriate combination of primers and template, and not with negative controls. The nucleotide sequence at the fusion site was confirmed by Sanger sequencing.

Interphase fluorescence in situ hybridization

A commercially available break-apart probe was used for ALK according to the manufacturer's protocol (Abbott, Des Plaines, IL). For ROS1, RET, NTRK1, and BRAF, break-apart fluorescence in situ hybridization (FISH) probes were prepared from BAC clones using standard procedures and labeled by nick translation with SpectrumGreen-dUTP and SpectrumRed-dUTP (Abbott, Des Plaines, IL). The following flanking BAC clones were used: RET (red: RP11-379D20, RP11-124O11; green: RP11-718J13, RP11-54P13), NTRK1 (red: RP11-284F21, RP11-66D17; green: RP11-1038N13, RP11-1059C21), ROS1 (red: RP11-379F24, RP11-103F10; green: RP11-1059G13, RP11-721K11), and BRAF (red: RP11-715H9, RP11-133N19; green: RP11-759K14, RP11-788O6). The probes were hybridized on 5μm-thick tissue sections, and the number and localization of the hybridization signals was assessed in a minimum of 100 interphase nuclei with well-delineated contours and at least 50% of neoplastic cells had to show a split signal to report a rearrangement of a kinase.

Immunohistochemistry

Immunohistochemical analysis was performed on archival FFPE tumor specimens to confirm the expression of the fusion kinases and the activation of oncogenic pathways. We used a Discovery Ultra instrument with a multimer/DAB detection system (Ventana Medical Systems, Inc., Tucson, AZ) with appropriate negative and positive controls the following antibodies: ALK (clone D5F3; Cell Signaling, Danvers, MA; Dilution: 1:250), ROS1 (clone D4D6; Cell Signaling, Danvers, MA; Dilution: 1:25), NTRK1 (clone EP1058Y; Epitomics, Burlingame, CA; Dilution: 1:100), RET (clone EPR287; Epitomics, Burlingame, CA; Dilution: 1:250). The percentage of tumor cells exhibiting staining was scored by at least two independent pathologists.

Plasmids

To investigate the functional roles and the activation of oncogenic signaling pathways, we cloned PWWP2A-ROS1, DCTN1-ALK, LMNA-NTRK1, and GOLGA5-RET fusions and expressed them in melan-a cells (immortalized, non-tumorigenic mouse melanocytes). Expression vectors with green-fluorescent-protein (GFP) and the full- length, wild-type kinases ROS1, ALK, NTRK1, and RET were used as controls. Plasmids containing cDNAs of the genes of interest were obtained (Supplementary Table S8) and their sequences were verified.^39-41 Non-synonymous mutations deviating from the RefSeq sequence found in the NTRK1 and RET plasmids were corrected using QuikChange site-directed mutagenesis (Agilent Technologies). Fusion constructs were generated by overlap extension.⁴² Once PCR products containing the target cDNAs were generated, they were cloned into a pENTR vector using the pENTR/D-TOPO cloning kit (Life Technologies). All constructs were subsequently cloned into the pLenti6.3/TO/V5-Dest backbone (Life Technologies) and checked by digestion and sequencing. The larger fusion genes involving ALK and ROS1 were also cloned into the PBX2.1 backbone of the PiggyBac Transposon system.⁴³

Stably transduced Melan-a cells

Melan-a cells were generously provided by Dr. Dorothy C Bennett (St. George's Hospital, University of London, London, UK)⁴⁴ and maintained in glutamine-containing RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum, 200 nM of 12-O-tetradecanoylphorbol-13-acetate (TPA), penicillin (100 units/mL) and streptomycin (50 mg/mL). 293FT cells were purchased from Life Technologies and maintained in DME-H21 medium containing 10% heat inactivated fetal bovine serum, MEM Non-Essential Amino Acids (0.1 mM), sodium pyruvate (1 mM), penicillin (100 units/mL) and streptomycin (50 mg/mL).

Lentiviruses were produced by transfecting 293FT cells with plasmid DNA in a pLenti6.3/TO/V5-Dest backbone together with ViraPowerTM (Life Technologies) according to the manufacturer's protocol. Infections of melan-a cells were conducted in the presence of 10μg/ml of Polybrene (Santa Cruz Biotechnology). Stably transduced melan-a cells expressing GOLGA5-RET, LMNA-NTRK1, PWWP2A-ROS1, RET, and NTRK1 were generated by infection with the respective lentivirus. ALK and ROS1 expressing melan-a cell lines were generated by cotransfection of the PBX2.1 construct together with pCMV-HyPBase transposase vector. Melan-a cell lines stably expressing GFP were generated using pLenti6.2/V5 and PBX2.1 vectors and used as a control. Cells were selected for at least 20 days using 5 μg/ml of blasticidin S-hydrochloride (Life Technologies) after transduction.

Western Blotting

Anti-RET (#3220), anti-phospho-RET (Tyr905) (#3221), anti-ALK (#3791), anti-phospho-ALK (Tyr1096) (#6962), anti-ROS1 (#3266), anti-phospho ROS1 (Tyr2274) (#3078), anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (#9101), anti-AKT (#9272), anti-phospho-AKT (Ser473) (#9271), anti-S6 Ribosomal Protein (#2317), anti-phospho-S6 Ribosomal Protein (Ser235/236) (#2211), anti-p-PLCγ1 (#2822), anti-phospho-PLCγ1 (Tyr783) (#2821), anti-SHP2 (D50F2) (#3397), anti-phospho-SHP2 (Tyr 542) (#3751), as well as the secondary antibodies anti-rabbit IgG-HRP (#7076) and anti-mouse IgG-HRP (#7074) were purchased from Cell Signaling Technology (Danvers, MA). Anti-HSP60 (sc-1722) and anti-ERK2 (C-14) (sc- 154) as well as the secondary antibodies anti-goat IgG-HRP (sc-2033) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-TrkA (ab37837) and anti-phospho-TrkA (Tyr408) (ab1445) were purchased from Abcam (Cambridge, MA).

Cell lysates were prepared in RIPA buffer supplemented with Halt protease and phosphatase inhibitor cocktail (Thermo Scientific). Equal amounts of protein, as measured by BCA protein assay, were resolved in 4-12% Bis-Tris NuPage gradient gels (Life Technologies) and transferred electrophoretically onto a PVDF 0.45-micron membrane. Membranes were blocked for 1 hour at room temperature in 5% BSA or non-fat milk in TBST before being incubated overnight at 4°C with the primary antibodies. All primary antibodies were diluted 1:1000 in 5% BSA or non-fat milk in TBST. After 3 washes of 5 minutes in TBST, secondary antibodies were diluted 1:3000 in 5% non-fat milk in TBST and incubated for 1 hour at room temperature. After another 3 washes in TBST, detection of the signal was achieved by incubating the membrane on an ECL solution from Millipore and exposure on autoradiography films from Denville Scientific (Metuchen, NJ).

Drug studies

Vandetanib and cabozantinib were purchased from Selleckchem (Houston, TX). Crizotinib was purchased from Chemie Tek (Indianapolis, IN). AZ-23 was purchased from Axon Medchem (Groningen, The Netherlands).

Tumorigenicity study

Animal experiments were carried out in accordance with the Declaration of Helsinki and were approved by the ethical committee of the University of California, San Francisco. 1.5 million melan-a cells stably transduced with the indicated oncogene or GFP control vector were injected subcutaneously in two separate, 6 week old female NOD-scid, IL2-Rγ^null mice. Mice were palpated 3 times a week for the development of tumors over a period of 40 days.