Clinicopathological findings of pediatric NTRK fusion mesenchymal tumors

Background While ETV6- NTRK3 fusion is common in infantile fibrosarcoma, NTRK1/3 fusion in pediatric tumors is scarce and, consequently, not well known. Herein, we evaluated for the presence of NTRK1/3 fusion in pediatric mesenchymal tumors, clinicopathologically and immunophenotypically. Methods We reviewed nine NTRK fusion-positive pediatric sarcomas confirmed by fluorescence in situ hybridization and/or next-generation sequencing from Seoul National University Hospital between 2002 and 2020. Results One case of TPR-NTRK1 fusion-positive intracranial, extra-axial, high-grade undifferentiated sarcoma (12-year-old boy), one case of LMNA-NTRK1 fusion-positive low-grade infantile fibrosarcoma of the forehead (3-year-old boy), one case of ETV6-NTRK3 fusion-positive inflammatory myofibroblastic tumor (IMT) (3-months-old girl), and six cases of ETV6-NTRK3 fusion-positive infantile fibrosarcoma (median age: 2.6 months, range: 1.6–5.6 months, M: F = 5:1) were reviewed. The Trk immunopositivity patterns were distinct, depending on what fusion genes were present. We observed nuclear positivity in TPR-NTRK1 fusion-positive sarcoma, nuclear membrane positivity in LMNA-NTRK1 fusion-positive sarcoma, and both cytoplasmic and nuclear positivity in ETV6-NTRK3 fusion-positive IMT and infantile fibrosarcomas. Also, the TPR-NTRK1 fusion-positive sarcoma showed robust positivity for CD34/nestin, and also showed high mitotic rate. The LMNA-NTRK1 fusion-positive sarcoma revealed CD34/S100 protein/nestin/CD10 coexpression, and a low mitotic rate. The IMT with ETV6-NTRK3 fusion expressed SMA. Six infantile fibrosarcomas with ETV6-NTRK3 fusion showed variable coexpression of nestin (6/6)/CD10 (4/5)/ S100 protein (3/6). Conclusions All cases of NTRK1 and NTRK3 fusion-positive pediatric tumors robustly expressed the Trk protein. A Trk immunopositive pattern and CD34/S100/nestin/CD10/SMA immunohistochemical expression may suggest the presence of NTRK fusion partner genes. LMNA-NTRK1 fusion sarcoma might be a low-grade subtype of infantile fibrosarcoma. Interestingly, more than half of the infantile fibrosarcoma cases were positive for S100 protein and CD10. The follow-up period of TPR-NTRK1 and LMNA-NTRK1 fusion-positive tumors are not enough to predict prognosis. However, ETV6-NTRK3 fusion-positive infantile fibrosarcomas showed an excellent prognosis with no evidence of disease for an average of 11.7 years, after gross total resection of the tumor.


Background
Next-generation sequencing (NGS) studies have recently revealed an increasing number of fusion genes in soft tissue sarcomas; these genes have been identified as oncogenic drivers and diagnostic markers of a wide range of adult and pediatric cancers [1]. However, until now, the clinicopathological characteristics of all these gene fusion tumors have not been clarified.
We have recently encountered pediatric cases of intracranial and forehead sarcomas. Pathologically, they did not fit into any known category of sarcomas or benign mesenchymal tumors. However, RNA sequencing by NGS of our cases revealed the presence of TPR-NTRK1, LMNA-NTRK1, and ETV6-NTRK3 fusions. Herein, we report these notable cases in detail so that their clinicopathological characteristics can be defined.

Patients
Nine pediatric NTRK fusion-positive sarcomas were retrieved from the archives of the Department of Pathology, Seoul National University Children's Hospital from 2002 to 2019. The fusion genes were detected by either fluorescence in situ hybridization (FISH) or NGS, such as RNA sequencing or customized gene panel study. One case of ETV6-NTRK3 fusion-positive IMT, one case of TPR-NTRK1 fusion-, one case of LMNA-NTRK1 fusion-and six cases of ETV6-NTRK3 fusion-positive sarcomas were reviewed.
Pathology, immunohistochemistry (IHC), and FISH All tumors were reviewed by two pathologists (JWK and SHP). IHC stains were performed on an immunostaining system (

DNA extraction and customized brain tumor gene panel study
On hematoxylin and eosin-stained FFPE sections, representative areas of tumors with at least 90% tumor cell purity were outlined for microdissection. DNA-extraction from the serial sections of the microdissected tumor tissue using the Maxwell® RSC DNA FFPE Kit (Promega, USA) was carried out according to the manufacturer's instructions.
The customized targeted gene panel (FIRST brain tumor panel and FIRST pan-cancer panel), which was customized and verified by the Department of Pathology of Seoul National University Hospital (SNUH), was used, containing 172 genes and ten fusion genes, and with a 1.7 Mb/run by NextSeq550Dx in Hi-Output. The produced sequencing data was analyzed using the pipeline of SNUH First Brain Tumor Panel Analysis. First, we performed the quality control of the Fastq file and analyzed only the data that passed the criteria. Pairedend alignment to the hg19 reference genome was performed using BWA-men and the GATK Best Practice [20]. After finishing the alignment step, an "analysisready BAM" was produced, and second quality control was performed to determine if further variant calling is appropriate. In the pipeline, single nucleotide variation (SNV), insertion and deletion (InDel), copy number variation (CNV), and translocation, were analyzed using at least more than two analysis tools, including in-house and open-source software. The open-source tools used were GATK UnifiedGenotyper, SNVer and LoFreq for SNV/InDel detection [21], Delly and Manta for Translocation discovery [22], THetA2 for purity estimation, and CNVKit for CNV calling [23], respectively. SnpEff was used to annotate the variants detected from various databases such as RefSeq, COSMIC, dbSNP, ClinVar, and gnomAD. The germline variant was then filtered using the population frequency of these databases (> 1% population frequency). Finally, the variants were confirmed through a comprehensive review of a multidisciplinary molecular tumor board.

RNA extraction, RNA sequencing, and fusion analysis
For RNA sequencing, the tumor RNA was extracted from the paraffin block (tumor fraction: > 90%) with Maxwell® RSC RNA FFPE Kit (Promega, USA). The library was generated with SureSelectXT RNA Direct Kit (Agilent, Santa Clara, USA), and sequenced on an Illumina NovaSeq 6000 at Macrogen (Seoul, Republic of Korea). Raw sequencing reads were analyzed with three kinds of algorithms, namely: DIFFUSE, Fusion catcher, and Arriba (https://github.com/suhrig/arriba/), to detect gene fusions. The results were then compared.
Fastq files were briefly aligned by the STAR aligner on the hg19 reference genome for Arriba analysis. The Fig. 1 a-e Case 1 with TPR-NTRK1 fusion.: MRI reveals a) T1-low, b-d) T2-high dura-based mass with enhancement. e The tumor was located in the right temporal convexity and right cerebellar tent (The direction of the brain mentioned as cephalhead and posterior). The inlet is the cut surface of the tumor showing yellowish, and solid without hemorrhage or necrosis. f, g Case 2 with LMNA-NTRK1 fusion tumor: T2 weighted MRI revealed low-density mass on the left forehead. The cut surface of the tumor shows a gray-white solid appearance without hemorrhage or necrosis chimeric alignments file and the read-through alignments file were produced, and fusion candidates were generated with a set of filters that detect artifacts based on various characteristic features.

Result
Clinicopathological findings and follow-up data of the patients The patient with TPR-NTRK1 fusion-positive sarcoma was a 12-year-old boy who presented with headache and diplopia for 3 months, and did not have any perinatal health problems. A 7.4-cm contrast-enhancing mass was detected in the right temporal lobe on magnetic resonance imaging (MRI) (Fig. 1a-d). Craniotomy revealed a hypervascular, extra-axial tumor with superficial brain invasion (Fig. 1e). Complete resection of the tumor with adjuvant chemotherapy with Ifosfamide, Carboplatin, and Etoposide (ICE) and radiation therapy (54 + 7.2 Gy) were administered because the pathology was high-grade undifferentiated sarcoma.
The patient with LMNA-NTRK1 fusion-positive sarcoma was a 3-year-old boy who presented with a growing mass on his left forehead, which had been present since he was a neonate as a pea-sized mass, and it has recently grown rapidly to 4.0 × 3.5 × 3.0 cm. It protruded from the forehead and was covered with eroded skin. The patient underwent complete surgical excision, and the cut surface of the tumor exhibited a homogenous tan-colored solid appearance ( Fig. 1f-g).
The patient with ETV6-NTRK3 fusion-positive IMT was a 3-month-old girl who presented with sudden onset dyspnea and systemic cyanosis. Chest computerized tomography (CT) showed a mass on the left lower thorax, that looked like a mass of the lower lobe of the left lung (Fig. 2). The mass was embolized under the impression of arteriovenous malformation at the local hospital. However, the symptom and signs were not relieved, and the mass had grown continuously to 5.6 × 5.2 × 3.3 cm. Lobectomy of the left lower lobe was then conducted to remove the tumor. Grossly, the mass was well-encapsulated and well-separated from the left lower lobe of the lung (Fig. 2). The tumor arose from an extrapulmonary sequestration, and was diagnosed as IMT by full pathological examination and NGS (using the customized First pan-cancer gene panel).
The median age of the six ETV6-NTRK3 fusion-positive infantile fibrosarcoma patients were 2.6 months (range: 1.6-5.6 months of age) at the time of surgery. The male to female ratio was 5: 1. The patients had presented with a mass on the tongue, buttock, right shoulder, left foot, right abdominal cavity, and sacrococcygeal area, respectively. Five tumors were completely resected, and adjuvant chemotherapies were given, as summarized in Table 1. The remaining massive sacrococcygeal tumor, involving the spinal cord, was initially subtotally resected and underwent three operations with one cycle of chemotherapy, but the patient was lost to follow-up.
The follow-up data are summarized in Table 1. The patients with TPR-NTRK1 and LMNA-NTRK1 fusion- positive sarcomas fared relatively well, with no tumor recurrence or neurological defects, during the 18 months and 11.6 months follow-up period, respectively. Five patients with ETV6-NTRK3 fusion-positive infantile sarcomas are all alive without disease for an average of 11.7 years (range: 6.0-17.4 years), but one case who had a huge sacrococcygeal mass was lost to follow-up.

Result of pathology, IHC, and FISH
Histopathology of the TPR-NTRK1 fusion-positive sarcoma showed a sheet of small oval-to-spindle cells with dilated blood vessels. Scanning power microscopy revealed a tiger-striped pattern due to vague layers of cellular and less-cellular areas with keloid type collagen deposits (Fig. 3). The tumor cells exhibited relatively uniform oval nuclei with fine chromatin and clear-to-eosinophilic cytoplasm. A high mitotic rate (25/10 per high-power fields) and a high Ki-67 labeling index (36.0%) were present; however, necrosis was not observed. The tumor cells were also robustly positive for Trk (1: 50, Cell Signaling, Boston, US), CD34, nestin, p53, and vimentin (Fig. 4). The robust nuclear positivity of Trk was remarkable (Fig. 5). However, the tumor cells were negative for S-100 protein, SMA, desmin, myogenin, CD99, Fli-1, CD56, STAT6, CK and EMA. TLE1 was weakly positive for the tumor cell nuclei and INI1 was retained.
RNA sequencing of an intracranial sarcoma (12-yearold boy) confirmed the presence of TPR-NTRK1 fusion   Fig. 1-4). Split reads are the read fragments of the unmatched paired-end alignments. A discordant alignment happens when both mates align uniquely, but does not satisfy the paired-end constraints.

Discussion
Primitive small round cell sarcomas and infantile fibrosarcomas are rare childhood sarcomas that pose diagnostic and therapeutic challenges. Recently, confirmative diagnosis of neoplasms has been made possible at the genomic level by identification of driver mutation or marker gene alterations [24]. Recent reports have described emerging pediatric fusion-positive sarcomas, including NTRK [5,8,25,26]. Our NTRK fusion-positive pediatric sarcomas have distinct immunohistochemical profiles. The TPR-NTRK1 fusion-positive tumor was a CD34-positive, dural-based, high-grade undifferentiated sarcoma with features that did not fit the classifications of existing types of sarcoma. In contrast, our LMNA-NTRK1 fusion-positive tumor was a low-grade spindle cell mesenchymal tumor of the forehead that was first noticed early in the neonatal period. The LMNA-NTRK1 fusion-positive tumor was difficult to diagnose before RNA sequencing by NGS because of its unusual pathology and immunohistochemical profile, namely, a combination of prominent inflammatory cells, no mitotic activity (0/10 HPF), and S100/CD34 coexpression. However, Hung et al.'s case of infantile fibrosarcoma also showed prominent inflammatory cells [4]. S100-protein and CD34 co-positivity are generally rare in sarcomas; these can be interpreted as hybridomas or evidence of dual differentiation. However, infantile fibrosarcomas often show coexpression of these two antibodies [16,27]. Miettinen et al. and Wong et al. reported a non-pleomorphic, low-grade spindle cell neoplasm with LMNA-NTRK1 fusion, that was diagnosed as infantile fibrosarcoma [17,27]. Miettinen et al. 's case showed low mitotic rates (< 5/10 HPFs), and S100 protein/CD34-coexpression [27]. Wong et al.'s case was CD34/vimentin-positive [17]. Our LMNA-NTRK1 fusion-positive sarcoma was consistent with Hung et al.'s and Miettinen et al.'s infantile fibrosarcoma with S100 protein/CD34 coexpression. The main differential diagnosis of this LMNA-NTRK1 fusion tumor was IMT because of prominent inflammatory cells in the tumor, but it can be ruled out based on its immunoprofile (SMA-negative, with S100/CD34 coexpression).
NTRK1 encodes TRKA receptor tyrosine kinase, which has a high affinity for nerve growth factor [3]. Genetic alterations of NTRK1 by translocations, amplifications, deletions, and point mutations have been reported in various tumor types, suggesting the potential role of Trk in oncogenesis [28,29]. More recently, NTRK1 chromosomal rearrangements have been identified in additional tumor types (Supplementary file, Table 2) [10, 21, 30,  31], suggesting that while oncogenic activation through NTRK1 fusion is not frequent, it can occur in various cancers. Interestingly, a significant number of NTRK1associated gene fusions have developed as a result of intrachromosomal gene fusion [11]. Depending on the directions of transcription of NTRK1 and its fusion partner, intrachromosomal fusions can occur either through simple interstitial deletion (e.g., LMNA-NTRK1) or through a more complex break/inversion mechanism (e.g., TPM3-NTRK1 or TPR-NTRK1), if the two genes are transcribed in opposite directions [13]. A 737-kbp deletion yielded the 5′ end of LMNA (localized to 1q22), including exons 1-10 fused to the 3′ end of NTRK1 (also localized to 1q22) and exons 12-17 [17].
Pan-Trk IHC can be used to detect NTRK fusion tumors; however, the expression site within the tumor cell differs according to the fusion partner genes [4]. We found strong nuclear envelope and cytoplasmic positivity in our LMNA-NTRK1 fusion-positive tumor. Intense nuclear staining in our TPR-NTRK1 fusion-positive sarcoma was observed with Trk (clone A7H6R) IHC, which is consistent with Hechtman et al. 's report using monoclonal antibody [MAb] EPR17341 [32]. However, a diffuse and strong cytoplasmic staining with MAb EPR17341 was reported in both LMNA-NTRK1 fusionpositive tumor and TPM3-NTRK1 fusion-positive sarcoma [1,33]. Davis et al. reported nuclear positivity in NTRK3 fusion tumors and cytoplasmic positivity in NTRK1/2 fusion tumors using the panTrk IHC (EPR17341) [8]. These differences in immunopositivity might be due to different Trk antibody clones and different types of sarcomas.
These NTRK fusion tumors tend to respond to NTRK inhibitors [2,11]. LOXO-101 is an orally bioavailable tyrosine kinase inhibitor that inhibits Trk catalytic activity with a low nanomolar potency. A phase 1 study with LOXO-101 in soft tissue sarcoma with LMNA-NTRK1 fusion and non-small cell lung cancer harboring TPR-NTRK1 fusion showed a good response [35,36]. Crizotinib was a durable response in the LMNA-NTRK1 fusion-positive undifferentiated pleomorphic sarcoma [37]. NTRK gene fusion could be a novel target of NTRK inhibitors for multiple tumor types [2].
In conclusion, we report two cases of NTRK1 fusionpositive and seven cases of NTRK3 fusion-positive pediatric sarcomas and IMT that were diagnostically challenging without molecular features. These three types of fusion-positive mesenchymal tumors (TPR-NTRK1, LMNA-NTRK1, and ETV6-NTRK3) differed in their H&E morphology, immunoprofile, and Trk immunopositivity patterns. In the case of LMNA-NTRK1 fusion sarcoma, S100/CD34/CD10-coexpression was a novel finding. The S100 protein, nestin, and CD10 positivity in infantile fibrosarcoma was also a new finding. The TPR-NTRK1 fusion sarcoma was positive for CD34 and nestin but negative for S100 protein. Thus, the Trk and CD34/S100/nestin/CD10 immunophenotype could be used for differential diagnosis. The sacrococcygeal infantile fibrosarcoma was unable to achieve complete resection, and the exact outcome is unknown because the patient was lost to follow-up. However, the remaining patients with ETV6-NTRK3 fusion-positive infantile fibrosarcomas survived for up to 17.3 years (median survival: 8.3 years), without tumor recurrence, after complete resection of the tumor. The patients with these fusion-positive tumors may benefit from NTRK inhibitor therapy if the tumors cannot be controlled by conventional treatment [38].