Spindle cell neoplasms with novel LTK fusion – Expanding the spectrum of kinase fusion‐positive soft tissue tumors

Kinase fusion‐positive soft tissue tumors represent an emerging, molecularly defined group of mesenchymal tumors with a wide morphologic spectrum and diverse activating kinases. Here, we present two cases of soft tissue tumors with novel LTK fusions.

spectrum of morphologies and histologic grades.At one end of this spectrum is the so-called lipofibromatosis-like neural tumor (LPF-NT), which is defined by haphazardly arranged monomorphic spindle cells, with tapering nuclei and indistinct cytoplasm. 2It shows a highly infiltrative pattern within subcutaneous fat, resembling lipofibromatosis.
Another morphologic subgroup is that reminiscent of low-to intermediate-grade malignant peripheral nerve sheath tumor (MPNST).
They are composed of monomorphic spindle cell proliferation arranged in short fascicles or patternless architecture.A diagnostic hallmark of this subset is the prominent stromal bands and perivascular rings of keloid-like hyalinized collagen. 3A few cases showing infiltrative growth within the fat-resembling LPF-NT have also been reported, suggesting a potential pathogenetic link between these two phenotypes.Specific soft tissue tumor entities include infantile fibrosarcoma (IFS) that harbors ETV6::NTRK3 fusion.It is composed of monomorphic spindle cells arranged in cellular sheets, fascicles, and/or herringbone patterns. 4A less common presentation of NTRK1-rearranged or RET-rearranged sarcoma includes tumors with a hemangiopericytic or myopericytoma-like pattern. 5Immunohistochemically, most tumors show coexpression of S100 and CD34, in the absence of SOX10 reactivity, whereas H3K27me3 expression is retained.The majority of tumors with NTRK fusions also express pan-TRK, which is a sensitive but non-specific marker of NTRK fusions among sarcomas. 6Recently, CLIP1::LTK fusions have been found in three cases of non-small cell carcinoma. 7However, LTK fusions in STTs have not been yet reported.
In this study, we report two cases of spindle cell tumors harboring the novel LTK fusion.We propose that they represent part of molecular spectrum of kinase fusion-positive soft tissue tumors.

| Case selection
The index case was found in Queen Mary Hospital, Hong Kong, during routine diagnostic RNA sequencing.Retrospective pathology database review was subsequently undertaken at Memorial Sloan Kettering Cancer Center, New York, USA, for additional case with the same gene fusion.
The study was approved by respective local Institutional Review Board.

| Targeted NGS DNA panel
DNA was extracted from FFPE, which is analyzed using ArcherDx VariantPlex custom panel targeting mutation hotspots of 97 genes and sequenced on Illumina sequencing platform.Variants are classified according to the AMP/ASCO/CAP standards and guidelines for interpretation and reporting sequence variants in cancer.

| Targeted RNA sequencing
RNA was extracted from the formalin-fixed, paraffin-embedded (FFPE) tissue.RNA was subjected to Illumina Pan-cancer RNA-sequencing panel (case 1).The library prepared was sequenced by NextSeq500/550 mid output v.2 kit at 76 bp pair-end on NextSeq500 sequencer (Illumina).The data were analyzed by RNA STAR Fusion pipeline.The raw reads were aligned to reference human genome (hg38) by STAR v.2.7.3. 8The fusion transcripts were determined by STAR Fusion software v.1.9 8and Arriba. 9Gene expression profiles will be determined and compared with cases with other kinase fusions, and other tumor types sequenced under the same panel, by using R bioconductor packages DESeq2. 10Targeted RNA sequencing for gene fusion detection in case 2 was performed by MSK-Fusion, a custom RNA-sequencing panel that uses the anchored multiplex PCR platform. 11,12Additionally, the probes on this assay target exons 1-13 of the LTK (NM_002344) transcript.

| DNA methylation-based classification
Genomic DNA was extracted from fresh-frozen section of the first case using DNeasy Blood & Tissue Kit (Qiagen).Methylation analysis was performed using Infinium MethylationEPIC V2 BeadChips (Illumina).Raw signal intensities were obtained from IDAT files of samples using the minfi Bioconductor package version 1.34.0. 13processed IDAT files from 1077 samples from the methylation profiling classifier developed by the German Cancer Research Center (Deutsches Krebsforschungszentrum, DKFZ) were used as a reference. 14Datasets of 1080 Illumina EPIC V2, EPIC V1, and 450 k samples were merged using the combineArrays function in minfi.After probe-filtering criteria were applied according to the GitHub repository, unsupervised non-linear dimension reduction was performed using the 10 000 most variable probes (according to mean absolute deviation) among combined 1080 samples.The t-distributed stochastic neighbor embedding (t-SNE) plots for the first case of LTK translocated tumor, two RAF1 translocated tumors and 1077 reference samples were made using the Rtsne package (version 0.15).Selective entities in differential diagnosis are chosen for hierarchical clustering and heatmap plotting using the R pheatmap package.The default agglomerative clustering method was applied on the calculated Euclidean distance among different entities.

| Clinical history
Index case 1 was that of a 17-year-old girl presenting with a progressively enlarging lesion over medial side of left big toe for 5 months.
There was no history of injury.Initial excision was performed with grossly involved margin.MRI was performed, showing a 2 Â 1.1 Â 1 cm solid mass lesion at antero-inferior aspect of left big toe pulp region of homogeneous signal intensity, being low/intermediate on T1 and mildly bright on T2.It caused focal bulging of left big toe pulp contours with no involvement of local musculotendinous, articular nor bony structures.PET scan was subsequently performed, revealing a 2-cm hypermetabolic soft tissue lesion at pulp of medial left big toe with SUVmax 4.8.No hypermetabolic nodal or distant metastasis was seen.Furthermore, excision with 1-cm margin was performed with wound left open.On pathologic examination, the tumor was marginally excised, with the deep margin being less than 0.1 mm.Second operation with completion amputation of distal phalangeal was performed, with no residual tumor noted.The patient was followed for 18 months, with no local recurrence or distant metastasis detected.
Case 2 was a 2-year-old male child presenting with swelling over ulnar aspect of left middle finger for a year.An MRI revealed a 2.5-cm contrast-enhancing fusiform mass involving the ulnar aspect of third proximal interphalangeal joint with high T2 signal.It contained strands of fat within it, accompanied by a 0.9-cm solid component and mild peritumoral soft tissue edema.No bone erosion was identified.The radiologic appearance was suggestive of granuloma annulare.
The tumor was subsequently removed in two fragments, and margins were involved.There was local recurrence 1 year later.Re-excision was performed showing a 3.5-cm lesion involving dermis and subcutis and extending to all margins.There was no evidence of disease after 10 years of follow-up.

| Histopathologic features and immunoprofile
For case 1, there was an infiltrative whitish lesion in the superficial dermis, extending to the subcutis (Figure 1A).around small vessels within the tumor nests.Scattered mitotic figures were identified, measuring at around 12 per 10 high-power fields.No coagulative tumor necrosis was seen (Figures 1B-G).
In case 2, the histology showed a low-cellularity spindle cell proliferation associated with abundant collagenous stroma infiltrating adipose tissue (Figure 2A).The pattern of invasion resembles that of pseudolipoblasts (Figure 2B).In areas, there was increased cellularity featuring uniform spindle cells arranged in streaming fascicles with open chromatin and eosinophilic cytoplasm (Figure 2C).Mitotic figures were inconspicuous.No necrosis was identified.The recurrent neoplasm demonstrated similar histologic features, with patchy increased cellularity involving subcutis (Figure 2D).Immunohistochemically, the tumor cells were focally positive for SMA, HHF35, and S100, but negative for CD34, AE1/3, EMA, desmin, and myogenin.
In case 2, targeted RNA-seq (Archer) identified MYH10::LTK fusion, with exon 35 of MYH10 gene (17p13.1;NM_005964) fused with exon 11 of LTK gene (15q15.1;NM_002344) (Figure 3D).Both fusions were in-frame, and the kinase domain of LTK was retained in both predicted chimeric proteins.None of these fusions appeared to be previously reported in any STTs in the literature, nor listed in the MSK cbioportal public database.Differential gene expression analysis in case 1 revealed that the LTK expression was higher than that a group of DFSP controls and other tumors sequenced by the same panel (Figures 3E).

| Multiomics analysis
Multiomics analysis was performed in case 1.In targeted DNA panel NGS, no clinically significant variants were detected.With t-SNE analysis using methylation profile, the index case 1 with LTK fusion, along with our previously reported case of RAF1 translocated tumor, 15   A diagnostic hallmark of this subset is the prominent stromal bands and perivascular rings of keloid-like hyalinized collagen. 3Both morphologic groups predominate in children and young adults, with LPF-NT typically occurring in the extremities, while the MPNST-like group may occur at various anatomic sites, including bone, soft tissue, and viscera.They usually display coexpression of CD34 and S100, with negative SOX10 staining and preserved H3K27me3 expression.Different kinase fusions have been implicated in the pathogenesis of STT, such as NTRK1, ALK, RAF1, RET, and BRAF; however, LTK fusion has not yet been reported in STT.Searching on public databases from TCGA 16 and MSK cbioportal 17 does not find any STT with LTK fusion.
In this study, we report novel LTK fusions in two soft tissue tumors.Both cases share similar clinicopathological features of occurring in acral sites of pediatric population.Moreover, both cases followed a good prognosis, given the radical-wide excision performed in both patients.The histology of both cases is consistent with the morphologic spectrum described for kinase fusion-positive spindle cell Leukocyte receptor tyrosine kinase, encoded by the LTK gene on chromosome 15q15.1, is a member of the ALK/LTK receptor family of receptor tyrosine kinases (RTKs).Following activation by binding ALKAL1 or ALKAL2 ligands to the extracellular domain, it induces tyrosine kinase activation, leading to activation of the PI3K/AKT and mitogen-activated protein kinase (MAPK) pathway involved in cellular growth and proliferation. 18,19The exact function of this protein is not known, but studies with chimeric proteins demonstrate its ability to promote growth and specifically neurite outgrowth, and cell survival.
It is also involved in regulation of the secretory pathway involving endoplasmic reticulum (ER), export sites (ERESs), and ER-to-Golgi transport. 20It is highly homologous to the anaplastic lymphoma kinase (ALK), with their cytoplasmic kinase domain being 79% identical.are involved in several important functions, including cytokinesis, cell motility, and maintenance of cell shape.[23][24][25] The fusion transcript involving translocation of MYH9 or MYH10 genes with LTK gene is expected to retain the 5 0 region of the former genes containing promoter regions and coiled-coil domain, and the 3 0 cytoplasmic kinase domain of LTK gene.Thus, it likely leads to increased expression of LTK, as seen in differential gene expression analysis.Phosphorylation of LTK protein with subsequent activation of downstream PI3K/AKT and MAPK pathways will contribute to increased cellular proliferation and tumorigenesis.
Interestingly, the DNA methylation clustering analysis suggests a closer histogenesis to DFSP, similar to our previously reported case of a primary bone sarcoma with MAP4::RAF1 fusion. 158][29][30] There are some morphological variants such as pigmented, myxoid, myoid, and fibrosarcomatous variants, 31 but the morphology of the current cases does not fully fit into classical or any of the known specific variant.The patchy coexpression of CD34 with S100 is also unusual.Therefore, despite the methylation profile, the morphology and immunoprofile of the LTK-positive tumors suggests a closer classification under the broad umbrella of molecularly defined category of kinase fusion-positive STTs.Intriguingly, our case was also close to a subset of MPNSTs by hierarchical clustering.This finding might be in line with the fact that a subset of kinase-rearranged STTs have a MPNST-like phenotype, which was the manifestation of the index case.Furthermore, study involving a larger number of cases will be warranted to fully elucidate the classification of tumors with LTK fusion.
Finally, current evidence indicates that many of these fusions lead to a state of oncogene addiction and, therefore, targeted therapies directed at constitutively activated oncogenic tyrosine kinases may prove to be a potentially viable treatment option for these fusionpositive sarcomas.A number of tyrosine kinase inhibitors are available in clinical trials or FDA-approved, such as cabozantinib, larotrectinib, and vemurafenib, respectively. 32The recently reported cases of nonsmall cell carcinoma with CLIP1::LTK fusion have demonstrated in vitro and in vivo efficacy by ALK inhibitors. 7 summary, we present two STTs with novel LTK fusions occurring at acral sites in pediatric patients, thus expanding the molecular spectrum of kinase fusion-positive soft tissue tumors.Regardless of its exact classification, recognizing these kinase fusion-positive STTs remains critical not only for diagnostic purposes, but also opens new avenues for targeted therapy.
The tumor was centered in the dermis with surface ulceration.It was composed of a monomorphic spindle cell proliferation of moderate cellularity.The cells were arranged in short fascicles, interconnecting nests or sheets with focal storiform-like pattern, accompanied by a hyalinized fibromyxoid stroma.There was wedge-shaped fat infiltration with focal suggestion of honeycomb entrapment.Mild perivascular hyalinization was noted F I G U R E 1 Histology and immunostainings of case 1 (A) Gross examination showed an infiltrative tumor in the dermis with surface epidermal ulceration.(B) At low power, the tumor was centered in the dermis, with (C) a wedge-shaped subcutis infiltration along the fibrous septa and very focal honeycomb pattern.(D) The spindle cells were arranged in short fascicles accompanied by a hyalinized stroma with stromal bands and (E) perivascular rings of keloid-like hyalinized collagen.(F) Areas of myxoid stroma were noted but no tumor necrosis was seen.(G) High-power view shows that the tumor cells have minimal pleomorphism with occasional mitotic figures.The tumor cells are patchily positive for (H) CD34 and (I) S100.
clustered with the DFSP in the DKFZ reference cohort (Figure 4A).By hierarchical clustering with a subset of tumors in differential diagnosis, such as DFSP and other nerve sheath tumors, the index case clustered with DFSP but also close to some MPNST (Figure 4B).Both LTK and F I G U R E 2 Histology of Case 2 (A) Low-power view showing a low-cellularity spindle cell proliferation associated with abundant collagenous stroma infiltrating adipose tissue (100Â) (B) Pattern of invasion into subcutaneous fat giving the impression of pseudolipoblasts (200Â) (C) High power within an area of increased cellularity showing uniform spindle cells arranged in streaming fascicles with open chromatin and eosinophilic cytoplasm (400Â).(D) The recurrent neoplasm showing similar histologic features, with patchy increased cellularity involving subcutis (200Â).RAF1 translocated tumors did not cluster together with other tumors harboring kinase fusion, like IFS and inflammatory myofibroblastic tumor (IMT).

4 |
DISCUSSIONWith the increasing clinical application of next-generation sequencing, many soft tissue tumors (STT) are found to be translocation driven.STT characterized by activating kinase fusions involving NTRK, ALK, RAF1 and RET genes among others, exhibit a wide morphologic spectrum.Except for a few well-established pathologic entities, such as IFS and IMT, many of these kinase fusion STTs were previously unclassified or misclassified under various diagnoses, and possibly represent novel and emerging tumor types.On one histological spectrum, it exhibits LPF-NT morphology defined by infiltrative growth into surrounding adipose tissues resembling lipofibromatosis.Another subset of cases resembles low-to intermediate-grade MPNST.They have a solid growth pattern, comprising a moderately to highly cellular proliferation of uniform spindle cells arranged in streaming or patternless patterns.

F
I G U R E 3 RNA-seq results.(A) Integrated Genome Viewer from RNA-seq featuring the MYH9::LTK fusion, which was confirmed by (B) RT-PCR with exon 34 of MYH9 fused with exon 11 of LTK.Schematic diagram of the (C) MYH9::LTK fusion and (D) MYH10::LTK fusion with retained kinase domain of LTK protein.(E) Differential gene expression analysis on case 1 showed that the expression of LTK was higher than that of DFSP and other tumors sequenced under the same panel.tumors.Specifically, both resemble LPF-NT featuring infiltrative growth of spindle cells into surrounding adipose tissues and patchy S100 expression.The first case may also show a hybrid morphology with "MPNST-like" solid areas, 2 depicting stromal bands and perivascular rings of keloid-like hyalinized collagen.

MYH9
gene and MYH10 gene are located on chromosome 22q12.3and 17p13.1,respectively.They encode myosin-9 protein and myosin heavy chain 10 as part of the non-muscle myosins.They F I G U R E 4 Methylation analysis.(A) t-sne analysis demonstrated the LTK translocated tumor and RAF1 translocated tumor clustered near DFSP among the DKFZ reference cohort.(B) By hierarchical clustering with cases in morphological differential diagnoses, it clustered close to DFSP and also some cases of MPNST.