Clinicopathological �ndings of Pediatric NTRK fusion mesenchymal tumors

Background: While ETV6-NTRK3 fusion is common in infantile �brosarcoma, NTRK1/3 fusion in pediatric tumors is scarce and, consequently, not well known. Herein, we evaluated NTRK1/3 fusion pediatric mesenchymal tumors clinicopathologically and immunophenotypically. Methods: We reviewed nine NTRK fusion pediatric sarcomas con�rmed by �uorescence 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 �brosarcoma of the forehead (3-year-old boy), one case of ETV6-NTRK3 fusion-positive in�ammatory myo�broblastic tumor (IMT) (3-months-old girl), and six cases of ETV6-NTRK3 fusion-positive infantile �brosarcoma (median age: 2.6 months, range: 1.6–5.6 months, M: F = 5:1) were reviewed. The Trk immunopositive pattern was distinctive according to the fusion genes. We noti�ed nuclear positivity in TPR-NTRK1 fusion sarcoma, nuclear membrane positivity in LMNA-NTRK1 fusion sarcoma, and both cytoplasmic and nuclear positivity in ETV6-NTRK3 fusion-positive IMT and infantile �brosarcomas. Also, the TPR-NTRK1 fusion sarcoma showed robust positivity for CD34/nestin and high mitoses. The LMNA-NTRK1 fusion sarcoma revealed CD34/S100 protein/nestin/CD10 coexpression, and a low mitotic rate. The IMT with ETV6-NTRK3 fusion expressed SMA. Six infantile �brosarcomas 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 phenotype may suggest NTRK fusion partner genes and diagnoses. LMNA-NTRK1 fusion sarcoma might be a low-grade subtype of infantile �brosarcoma. Interestingly, more than half of the infantile �brosarcoma cases were positive for S100 protein and CD10. The follow-up period of TPR-NTRK1 and LMNA-NTRK1 fusion tumors are not enough to predict prognosis. However, ETV6-NTRK3 fusion infantile �brosarcomas 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 identi ed 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 clari ed.
We have recently encountered pediatric cases of intracranial and forehead sarcomas.Pathologically, they did not t 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 de ned.

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 uorescence 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.

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.DNAextraction 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 veri ed 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 le and analyzed only the data that passed the criteria.Paired-end alignment to the HG19 reference genome was performed using BWA-men and the GATK Best Practice [20].After nishing the alignment step, an "analysis-ready 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 Uni edGenotyper, 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 ltered using the population frequency of these databases (> 1% population frequency).Finally, the variants were con rmed 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 para n 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 les were brie y aligned by the STAR aligner on the hg19 reference genome for Arriba analysis.The chimeric alignments le and the read-through alignments le were produced, and fusion candidates were generated with a set of lters that detect artifacts based on various characteristic features.

Clinicopathological ndings 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 three 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. 1).

Craniotomy revealed a hypervascular, extra-axial tumor with super cial brain invasion. Complete resection of the tumor with adjuvant chemotherapy with
Ifosfamide, Carboplatin, and Etoposide (ICE) and radiation therapy (54+7.2Gy) 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. 1).
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 5.6x5.2x3.3cm.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 brosarcoma 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: 6.0-17.4years), 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 ne chromatin and clear-to-eosinophilic cytoplasm.A high mitotic rate (25/10 per high-power elds) 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.

Discussion
Primitive small round cell sarcomas and infantile brosarcomas are rare childhood sarcomas that pose diagnostic and therapeutic challenges.Recently, con rmative diagnosis of neoplasms has been made possible at the genomic level by identi cation 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 pro les.The TPR-NTRK1 fusion-positive tumor was a CD34-positive, dural-based, high-grade undifferentiated sarcoma with features that did not t the classi cations 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 rst noticed early in the neonatal period.The LMNA-NTRK1 fusion-positive tumor was di cult to diagnose before RNA sequencing by NGS because of its unusual pathology and immunohistochemical pro le, namely, a combination of prominent in ammatory cells, no mitotic activity (0/10 HPF), and S100/CD34 coexpression.However, Hung et al.'s case of infantile brosarcoma also showed prominent in ammatory cells [4].S100protein and CD34 co-positivity are generally rare in sarcomas; these can be interpreted as hybridomas or evidence of dual differentiation.However, infantile brosarcomas 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 brosarcoma [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 brosarcoma with S100 protein/CD34 coexpression.The main differential diagnosis of this LMNA-NTRK1 fusion tumor was IMT, but it can be ruled out based on its immunopro le (SMA-negative, with S100/CD34 coexpression).
NTRK1 encodes TRKA receptor tyrosine kinase, which has a high a nity for nerve growth factor [3]. Genetic alterations of NTRK1 by translocations, ampli cations, 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 identi ed in additional tumor types (Supplementary le, 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 signi cant number of NTRK1-associated 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 fusionpositive 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 fusion-positive 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.
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 fusion-positive 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, immunopro le, and Trk immunopositivity patterns.In the case of LMNA-NTRK1 fusion sarcoma, S100/CD34/CD10-coexpression was a novel nding.The S100 protein, nestin, and CD10 positivity in infantile brosarcoma was also a new nding.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 brosarcoma was unable to achieve complete resection, and the exact outcome is unknown because the patient was lost to followup.However, the remaining patients with ETV6-NTRK3 fusion-positive infantile brosarcomas 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 bene t from NTRK inhibitor therapy if the tumors cannot be controlled by conventional treatment [38].Consent for publication: All materials had been obtained for medical care of the patients, which were anonymized and retrospectively reviewed.No extrahuman materials were obtained for this study.Under the Korean Bioethics and Safety Act, additional consent to publish was waivered.

Figures Figure 1 A
Figures

Figure 2 A
Figure 2 A) Chest CT of the ETV6-NTRK3 fusion-positive in ammatory myo broblastic tumor (IMT) reveals a heterogeneously enhancing tumor in the left lower part of the thorax.B) The mass arises from extrapulmonary sequestration, supplied by the left phrenic artery, which is separated from the lower lobe of the lung.C) The cut surface of the tumor is hemorrhagic and has congested large vessels.D) This is the microscopic picture of the squared part of gure C. It is a wellencapsulated, but partly adhered to the lower lobe of the left lung.The tumor arose from the extrapulmonary sequestration and pushed the left lower lobe of the lung.Hemorrhage was developed by previous embolization of large vessels of the sequestrated lung, under the impression of arteriovenous malformation.

Figure 3 A
Figure 3 A) Histology of the intracranial undifferentiated sarcoma with TPR-NTRK1 fusion shows alternating cellular areas with collagen bands show a tiger-like appearance.The tumor cells are oval to short spindle cells.B) The forehead mesenchymal tumor with LMNA-NTRK1 fusion shows relatively low cellular spindle cell mesenchymal tumor with keloid type collagen laydown.C) The pulmonary in ammatory myo broblastic tumor with ETV6-NTRK3 fusion shows bland-looking spindle cells with intermixed lymphoplasma cells.D) A sacrococcygeal Infantile brosarcoma with ETV6-NTRK3 fusion shows fascicular spindle cells with high cellularity (A-D: H&E, bar: A-D: 50μm).

Figure 5 Trk
Figure 5 Trk immunohistochemistry shows A) Nuclear positivity in TPR-NTRK1 fusion sarcoma (Case 1), B) mostly nuclear membrane, and cytoplasmic stain in LMNA-NTRK1 fusion sarcoma (Case 2).and C) mainly cytoplasmic stain in ETV6-NTRK3 fusion sarcoma.In gure B, the Trk-negative cells are in ltrating in ammatory cells (A-C: Trk IHC, lower bar: 50 μm).D. locus-speci c identi er (LSI) FISH study using ETV6 uorescence dual-color break apart DNA probes show one fused and one widely separated SpectrumGreen and SpectrumOrange signals in an infantile brosarcoma with ETV6-NTRK3 fusion-positive.