Pediatric NTRK fusion sarcomas

Background While ETV6-NTRK3 fusion is common in infantile �brosarcoma, NTRK1 fusion in pediatric sarcoma is extremely rare and consequently not well known. Herein, we evaluated NTRK1 fusion pediatric sarcomas to determine their clinicopathological nature and differential diagnosis from classic ETV6-NTRK3 fusion infantile �brosarcoma. Methods


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 of these gene fusion tumors have not been clari ed.

Patients
Eight NTRK fusion sarcomas were retrieved from the archives of pediatric sarcomas under 18-year-old from the archives of the Department of Pathology, Seoul National University Children's Hospital from 2002 to 2019.Among them, authors reviewed clinicopathological and genetic ndings of 8 NTRK fusionpositive sarcomas, detected by either uorescence in situ hybridization (FISH) or next-generation sequencings (NGS), such as RNA sequencing or customized gene panel study.They were one case of TPR-NTRK1 fusion-, one case of LMNA-NTRK1 fusion-and six cases of ETV6-NTRK3 fusion-sarcomas.Their clinicopathologic ndings are summarized in Table 1.

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 targeted gene panel (FIRST brain tumor panel), which was customized and veri ed by the Department of Pathology of SNUH, containing 172 genes and ten fusion genes, 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 HG19 reference genome was performed using BWA-men and the GATK Best Practice. 16After 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, SNV, InDel, 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 17 , Delly and Manta for Translocation discovery 18 , THetA2 for purity estimation, and CNVKit for CNV calling 19 , respectively.SnpEff annotated detected variants with various databases such as RefSeq, COSMIC, dbSNP, ClinVar, and gnomAD.Then germline variant was ltered using the population frequency of these databases (> 1% population frequency).Finally, the variants were con rmed throughout 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 of DIFFUSE, Fusion catcher, and Arriba (https://github.com/suhrig/arriba/) to detect gene fusions, and compared the results.
Brie y, Fastq les were 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.

Result Clinicopathological ndings and follow-up data of the patients
The patient with TPR-NTRK1 fusion sarcoma was a 12-year-old boy presented with headache and diplopia for three months, who 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 + 7.2 Gy) were administered because the pathology was high-grade undifferentiated sarcoma.
One patient with LMNA-NTRK1 fusion sarcoma was a 3-year-old boy who presented with a growing mass on his left forehead, which had been present since his neonatal periord as pea size, 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 patients' median ages of six ETV6-NTRK3 fusion-positive infantile brosarcomas at the time of surgery were 2.6 months (range: 1.6-5.6 months of age).
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, which are summarized in Table 1.The remaining massive sacrococcygeal tumor, involving the spinal cord, was initially subtotally resected and underwent three times of operation with one cycle of chemotherapy, but the follow-up of the patient was lost.
These follow-up data were also summarized in Table 1.The patients with TPR-NTRK1 fusion and LMNA-NTRK1 sarcomas fared relatively well for 18 months and 11.6 months follow-up period, without tumor recurrence or neurological defects.Six cases of ETV6-NTRK3 fusion sarcomas are all alive without disease for an average of 11.7 years (range: 6.0-17.4years) except one case who had a huge sacrococcygeal mass and lost follow-up.

Result of pathology, immunohistochemistry (IHC), and uorescence in situ hybridization
Histopathology of TPR-NTRK1 fusion 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. 2).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, and vimentin (Fig. 2).The robust nuclear positivity of Trk was remarkable (Fig. 3).However, the tumor cells were negative for S-100 protein, SMA, desmin, myogenin, CD99, Fli-1, CD56, STAT6, p53, cytokeratin, and EMA.TLE1 was weakly positive for the tumor cell nuclei.INI1 was retained.
LMNA-NTRK1 fusion tumor was composed of vaguely fascicular spindle cells with bland-looking elongated nuclei and inconspicuous nucleoli (Fig. 2).There was collagen lay down between the tumor cells.Intermixed in ammatory cell in ltration was remarkable, which was pronounced on CD3 IHC (Fig. 2).The Ki-67 index was moderately high (18.2%),but many of them might be in ltrated in ammatory cells.Mitosis was absent on pHH3 IHC.There was neither necrosis nor hemorrhage.Therefore, this tumor was much less cellular and much more bland-looking than TPR-NTRK1 or ETV6-NTRK3 fusion sarcoma.The tumor cells were robustly and diffusely positive for Trk, S100-protein, CD34, and nestin (Fig. 2).The nuclear envelope-positivity for Trk was remarkable with weak cytoplasmic staining (Fig. 3).

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. 20Recent reports have described emerging pediatric fusion-positive sarcomas. 5,21,22 Or pediatric sarcomas had 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, extremely low mitotic activity (nearly 0/10 HPF), and S100/CD34 coexpression.However, Hung et al.'s case of infantile brosarcoma also showed prominent in ammatory cells. 4S100-protein and CD34 copositivity are generally rare in sarcomas, which can be interpreted as hybridomas or dual differentiation; however, infantile brosarcomas often show coexpression of these two antibodies. 13,23 iettinen et al. reported that non-pleomorphic, low-grade spindle cell features characterized LMNA-NTRK1 fusion infantile brosarcomas, low mitotic rates (< 5/10 HPFs), and S100 protein-positivity with often CD34-coexpression. 23Our LMNA-NTRK1 fusion sarcoma was consistent with Hung et al.'s and Miettinen et al.'s S100/CD34 coexpressing infantile brosarcomas.NTRK1 encodes TRKA receptor tyrosine kinase, which has a high a nity for nerve growth factor. 3Genetic 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. 24,25 ore recently, NTRK1 chromosomal rearrangements have been identi ed in additional tumor types (Supplementary le, Table 2), 7,17,26,27 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. 8Depending 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. 10A 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. 14n-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. 4Our LMNA-NTRK1 fusion tumor cells showed strong nuclear envelop and cytoplasmic staining.In contrast, our TPR-NTRK1 fusion sarcoma revealed strong nuclear staining for Trk (clone A7H6R), consistent with Hechtman et al.'s report using the same clone (monoclonal antibody [Mab] EPR17341).However, Rudzinski et al. reported that the LMNA-NTRK1 fusion sarcoma in their study showed diffuse and strong cytoplasmic staining with the Trk antibody (MAb EPR17341).In contrast, TPM3-NTRK1 fusion tumors showed strong and diffuse cytoplasmic staining. 1Thus, these differences might be due to various Trk antibody clones.
ETV6-NTRK3 and (rarely) EML4-NTRK3, LMNA-NTRK1, TPM3-NTRK1, and SQSTM1-NTRK1 fusions have been reported in infantile brosarcoma (Table 1). 4,13 e six cases of classic infantile brosarcoma in our study had an ETV6-NTRK3 fusion veri ed by ETV6 break-apart FISH and/or RNA sequencing.The diffuse cytoplasmic Trk positivity in our cases is consistent with that in previous reports of Trk immunopositivity patterns. 4,23 se NTRK fusion tumors tend to respond to NTRK inhibitors. 2,8 OXO-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 non-small cell lung cancer harboring TPR-NTRK1 fusion showed good response. 28NTRK gene fusion could be a novel target of NTRK inhibitors for multiple tumor types. 2 In conclusion, we report two cases of NTRK1 fusion and six cases of NTRK3 fusion pediatric sarcomas that were diagnostically challenging before NGS or FISH; the three types of fusion sarcomas (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.While the nding of S100 protein, nestin, and CD10 positivity in infantile brosarcoma is new, the TPR-NTRK1 fusion sarcoma was negative for S100 protein.The most interesting ndings were the distinctive Trk immunopositivity patterns according to the fusion subtype and that the CD34/S100/nestin/CD10 immunopro le could be used to suggest the NTRK fusion partner genes.The patients with NTRK fusion infantile sarcomas survived for up to 17.3 years without tumor recurrence except for a sacrococcygeal tumor, regardless of the histological grade, cellularity, or ki-67 labeling index.The median survival of our infantile brosarcomas was 8.3 years.Furthermore, patients with these fusion tumors may bene t from NTRK inhibitor therapy if the tumors cannot be controlled by conventional therapy. 29st Of Abbreviations 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.
Availability of data and materials: All the genetic data can be found in our SNUH's big data server managing by the center for precision medicine.

Figures
Figures

Figure 1 A
Figure 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 inlet is the cut surface of the tumor, 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.

Figure 2 A
Figure 2 A, B) Intracranial sarcoma with TPR-NTRK1 fusion.A) Variably cellular and less cellular pattern with Keloid type brosis.The tumor cells are oval to short spindle cells.B) The alternating cellular areas with collagen bands show a tiger-like appearance.C) The forehead mesenchymal tumor with LMNA-NTRK1 fusion shows relatively low cellular spindle cell mesenchymal tumor with keloid type collagen laydown.D) A sacrococcygeal Infantile brosarcoma shows fascicular spindle cells with high cellularity (A-D: H&E, bar: A, B, C-D: 500, 300, 200 micrometers).

Figure 4 Trk
Figure 4 Trk immunohistochemistry shows A) Nuclear positivity in TPR-NTRK1 fusion sarcoma (Case 1), B) mostly nuclear membrane, and some 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, B, C: Trk IHC, bar: A, B: 200 micrometers, C: lower bar: 100 micrometers).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.