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 findings of 8 NTRK fusion-positive sarcomas, detected by either fluorescence 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 findings are summarized in Table 1.
Table 1
Clinicopathological comparison of our two cases of TPR-NTRK1 and LAMA-NTRK1 fusion-positive sarcomas and 5 cases of our infantile fibrosarcoma.
| TPR-NTRK1 fusion-positive pediatric sarcoma | LMNA-NTRK1 fusion-positive pediatric sarcoma | ETV6-NTRK3 fusion-positive pediatric sarcoma |
Age/Gender | 12 y/ male | 3 y/ male | Median age: 3.1 months (range: 1.6 ~ 4.0 months), M: F = 5:1 |
Site | Dura, parieto-occipital | Forehead dermis and subcutaneous tissue | Tongue, buttock, shoulder, foot, abdominal cavity, and sacrococcygeal area |
Size | 6.0 × 5.0 × 3.0 cm | 4.0 × 3.5 × 3.0 cm | Range: 1.4–4.5 cm in biggest diamenter |
Histology | Mixed round cell and spindle cell | Spindle cells intermixed with prominent inflammatory cells | Mostly spindle cell, with no prominent inflammatory cell infiltration |
Histological grade | High-grade | Low-grade | High-grade (cellular) |
Nuclear pleomorphism | Uniform cell | Uniform cell | Uniform cell |
Tumor necrosis | Absent | Absent | Absent |
Mitotic rate | 20/10 HPFs | 0/10 HPFs | 5/10HPFs ~ 40/10 HPFs |
Ki67 labeling index | 22.5% | 18.2% | 15 ~ 60% |
Immuno-positive markers | Trk+/Nestin+/vimentin+/CD34+/p53 | Trk+/Nestin+/vimentin+/S100+/CD34+ | Trk+/Nestin/vimentin+, S100+ (3/6 cases)/CD10+(4/5 cases) |
Trk positive pattern | Nuclear positive | Nuclear membrane-positive | Cytoplasmic and nuclear positive |
Immuno-negative markers | S100-/CD56-/SMA-/desmin-/myogenin-/STAT6-/EMA-/CK-/ERG-/CD99-/CD21/CD35-/GFAP-/Olig2-/p16+/INI-1+ | CD56-/SMA-/desmin-/myogenin-/STAT6-/EMA-/CK-/CD1a-/CD21-/CD35-/CD43-/WT-1-/MelanA-/HMB45-/BRAF-/ALK- | CD34-/myogenin-/desmin |
Other genetic abnormalities | NTRK1 amplification (copy number: 11), H3F3A amplification (copy number: 12) | absent | Absent |
Final diagnosis | Undifferentiated sarcoma | Infantile fibrosarcoma, low grade (according to previous report by et al.) | Infantile fibrosarcoma, cellular |
known mesenchymal tumors with this fusion | Lipofibromatosis, adult uterine undifferentiated sarcoma | Low-grade infantile fibrosarcoma, lipofibromatosis-like neural tumor, Undifferentiated sarcoma, cellular meoblastic nephroma | Infantile fibrosarcoma |
Known gene fusions of infantile fibrosarcoma | | | ETV6-NTRK3, EML4-NTRK3, LMNA-NTRK1, TPM3-NTRK1, SQSTM1-NTRK1(1, 2) |
1. Hung YP, Fletcher CDM, Hornick JL. Evaluation of pan-TRK immunohistochemistry in infantile fibrosarcoma, lipofibromatosis-like neural tumour and histological mimics. Histopathology. 2018;73:634–644. |
2. Davis JL, Lockwood CM, Albert CM, et al. Infantile NTRK-associated Mesenchymal Tumors. Pediatr Dev Pathol. 2018;21:68–78. |
Pathology, immunohistochemistry (IHC), and fluorescence in situ hybridization-study
All tumors were reviewed by two pathologists (JWK and SHP). Immunohistochemical (IHC) stain performed on an immunostaining system (BenchMark ULTRA system, Ventana-Roche, Mannheim, Germany) using primary antibodies including Trk (1: 50, Cell signaling, Boston, USA), nestin (1: 200, Millipore, Temecula, USA), vimentin (1: 500, DAKO, Grostrup, Denmark), S100 protein (1: 3000, DAKO), CD34 (1: 200, Dako), CD10 (RTU, Novocastra, Newcastle, UK), Ki67 (1: 100, MAb MIB-1; Dako), and phosphohistone-H3 (1: 5000, Cell Marque, Rocklin, USA), TLE1 (1: 20, Cell Marque, Rocklin, US), Fli1 (1: 300, Becton and Dickinson, Flanklin Lakes, US), p53 (1: 100, DAKO), ERG (rtu, Ventana, Export, US), CD99 (1: 200, Novocastra (Leica), Muchen, Germany), Smooth muscle actin (SMA, 1: 500, DAKO), Desmin (1:200, DAKO), Myogenin (1: 500, DAKO), cytokeratin (1: 300, DAKO), epithelial membrane antigen (EMA, 1: 300, DAKO), Integrase interactor 1 (INI-1, 1: 100, Cell signaling,), STAT6 (1: 100, ABCAM, Cambridge, UK) (Suppelementary file: Table 1). Appropriate positive controls were included, and for the negative control, primary antibodies were omitted. Mitotic activity was assessed with pHH3 immunostain on 4 µm thick FFPE slides by counting mitotic figures in 10 high power fields (HPF; area, 2.38 mm2). The immunohistochemical antibodies that were used are summarized in Supplementary file, Table 1.
For ETV6 break-apart FISH study, locus-specific identifier (LSI) Vysis ETV6 fluorescence dual-color break apart DNA probes, ETV6 (CEN) SpectrumGreen and Vysis LSI ETV6 (TEL) SpectrumOrange (Abbott Molecular, Abbott Park, US), was used.
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 targeted gene panel (FIRST brain tumor panel), which was customized and verified 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 file 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.16 After finishing 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 UnifiedGenotyper, SNVer, and LoFreq for SNV/InDel detection17, Delly and Manta for Translocation discovery18, THetA2 for purity estimation, and CNVKit for CNV calling19, respectively. SnpEff annotated detected variants with various databases such as RefSeq, COSMIC, dbSNP, ClinVar, and gnomAD. Then germline variant was filtered using the population frequency of these databases (> 1% population frequency). Finally, the variants were confirmed 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 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 of DIFFUSE, Fusion catcher, and Arriba (https://github.com/suhrig/arriba/) to detect gene fusions, and compared the results.
Briefly, Fastq files were aligned by the STAR aligner on the hg19 reference genome for Arriba analysis. The 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 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 superficial 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 fibrosarcomas 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: 6.0-17.4 years) except one case who had a huge sacrococcygeal mass and lost follow-up.
Result of pathology, immunohistochemistry (IHC), and fluorescence 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 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, 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 inflammatory cell infiltration 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 infiltrated inflammatory 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).
Histopathology of ETV6-NTRK3 fusion sarcomas showed highly cellular and relatively uniform small spindle cells with a high mitotic rate (10–40/10 HPFs). There were neither necrosis nor prominent inflammatory cell infiltration in all cases. These infantile fibrosarcomas were diffusely and robustly positive for Trk (100%), S100 protein (50%, 3/6 cases), nestin, CD10 (80%, 4/5 cases), and vimentin (100%), but negative for CD34, SMA, desmin, myogenin, and CD56. The Trk IHC showed a diffuse cytoplasmic stain with some nuclear staining (Fig. 3). Ki-67 labeling indices were 15–60%. ETV6-NTRK3 fusion was verified by fluorescence in situ hybridization in all six cases (Fig. 3) and additionally by RNA sequencing in two cases.
The targeted gene panel revealed a TPR-NTRK1 fusion of TPR on chromosome 1q25 (position 186337018) and NTRK1 on chromosome 1q21-q22 (position 156844363) with amplification of NTRK1 and H3F3A on chromosome 1 in case 1 (Fig. 4).
RNA sequencing of an intracranial sarcoma (12-year-old boy) confirmed TPR-NTRK1 fusion (Breakpoint: 1: 186337018, 1: 156844363), and a forehead tumor (3-year-old boy) resulted in LMNA-NTRK1 fusion (Breakpoint: 1: 156104766, 1: 156844698). The number of split reads in TPR and NTRK1 was 35 and 31, respectively, and there were two discordant mates, and 37 split reads in LMNA and 53 in NTRK1, with seven discordant mates. Two cases of infantile fibrosarcomas performed RNA sequencing showed ETV6-NTRK3 fusion (Case 4: Breakpoints: and the split reads of ETV6 and NTRK3 (Breakpoints: 12: 12022903: 15: 88483984, 12: 12022903, 15: 88524591) were 11 and 16, and 25 and 8, respectively (Supplementary Fig. 1–4). Split reads are the reads fragments of the unmatched paired-end alignments. A discordant alignment is an alignment where both mates align uniquely, but that does not satisfy the paired-end constraints.