Recurrent EML4–NTRK3 Fusions in Infantile Fibrosarcoma And

Modern Pathology (2018) 31, 463–473

Recurrent EML4–NTRK3 fusions in infantile fibrosarcoma and congenital mesoblastic nephroma suggest a revised testing strategy Alanna J Church1, Monica L Calicchio2, Valentina Nardi3, Alena Skalova4, Andre Pinto5, Deborah A Dillon6, Carmen R Gomez-Fernandez5, Namitha Manoj7, Josh D Haimes7, Joshua A Stahl7, Filemon S Dela Cruz8, Sarah Tannenbaum-Dvir9, Julia L Glade-Bender9, Andrew L Kung7, Steven G DuBois10, Harry P Kozakewich1, Katherine A Janeway10, Antonio R Perez-Atayde1 and Marian H Harris1

1Department of Pathology, Boston Children’s Hospital and Harvard Medical School, Boston, MA, USA; 2Department of Pathology, Boston Children’s Hospital, Boston, MA, USA; 3Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA; 4Department of Pathology, Charles University, Faculty of Medicine in Plzen, Plzen, Czech Republic; 5Department of Pathology, University of Miami, Miami, FL, USA; 6Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA; 7ArcherDX, Boulder, CO, USA; 8Department of Pediatrics, Memorial Sloan Kettering Cancer Institute, New York, NY, USA; 9Division of Pediatric Hematology, Oncology and Stem Cell Transplantation, Columbia University Medical Center, New York, NY, USA and 10Department of Pediatric Hematology/Oncology, Dana-Farber/Boston Children’s Cancer and Blood Disorders Center and Harvard Medical School, Boston, MA, USA

Infantile fibrosarcoma and congenital mesoblastic nephroma are tumors of infancy traditionally associated with the ETV6–NTRK3 gene fusion. However, a number of case reports have identified variant fusions in these tumors. In order to assess the frequency of variant NTRK3 fusions, and in particular whether the recently identified EML4–NTRK3 fusion is recurrent, 63 archival cases of infantile fibrosarcoma, congenital mesoblastic nephroma, mammary analog secretory carcinoma and secretory breast carcinoma (tumor types that are known to carry recurrent ETV6–NTRK3 fusions) were tested with NTRK3 break-apart FISH, EML4–NTRK3 dual fusion FISH, and targeted RNA sequencing. The EML4–NTRK3 fusion was identified in two cases of infantile fibrosarcoma (one of which was previously described), and in one case of congenital mesoblastic nephroma, demonstrating that the EML4–NTRK3 fusion is a recurrent genetic event in these related tumors. The growing spectrum of gene fusions associated with infantile fibrosarcoma and congenital mesoblastic nephroma along with the recent availability of targeted therapies directed toward inhibition of NTRK signaling argue for alternate testing strategies beyond ETV6 break-apart FISH. The use of either NTRK3 FISH or next-generation sequencing will expand the number of cases in which an oncogenic fusion is identified and facilitate optimal diagnosis and treatment for patients. Modern Pathology (2018) 31, 463–473; doi:10.1038/modpathol.2017.127; published online 3 November 2017

Infantile fibrosarcoma (also known as congenital cases of infantile fibrosarcoma are diagnosed during fibrosarcoma) is a malignant tumor of infancy.1 The the first year of life, although tumors have occasion- classic presentation is a rapidly enlarging mass on ally been detected in utero.2,3 The histopathology the extremity of a child under 2 years of age. Most varies greatly, and the differential diagnosis is broad. The classic form is composed of long fascicles of densely cellular spindle cells with a variable amount Correspondence: Dr MH Harris, MD, PhD, Laboratory for Mole- of collagenous stroma. On the other side of the cular Pediatric Pathology, Department of Pathology, Boston morphologic spectrum, infantile fibrosarcoma can be Children’s Hospital, 300 Longwood Avenue, Boston, MA 02115, organoid, with a hemangiopericytoma-like vascular USA. E-mail: [email protected] pattern, and plump cells with ovoid nuclei, more Received 6 June 2017; revised 13 August 2017; accepted 17 August rounded cytoplasm, and scant poorly collagenous 2017; published online 3 November 2017 stroma. This latter appearance may be www.modernpathology.org Recurrent EML4–NTRK3 in IFS and CMN 464 AJ Church et al

indistinguishable from infantile hemangiopericy- secretory carcinoma.30,31 In the past, such novel toma, a distinct morphologic variant of infantile fusions may have been of more academic than myofibroma/myofibromatosis. Mitoses, apoptosis practical interest. Today, however, their identifica- and areas of necrosis are frequently present in tion carries significant therapeutic implications with infantile fibrosarcoma, but again, these features are recent reports describing treatment of infantile variable and zonal. Not uncommonly, it has a fibrosarcoma with targeted therapy. One report mononuclear inflammatory infiltrate scattered describes treating a patient with infantile fibrosar- throughout the tumor. The clinical outcome is coma with a targeted inhibitor of the tropomyosin- generally favorable, with rare metastases and a related kinase (TRK) family, of which NTRK3 is one mortality rate of o5%, although tumors recur in member, as part of a Phase 1 trial of the drug.32 The up to 50% of patients and patients typically require a completed trial showed a 91% objective response combination of chemotherapy and surgery.1,3–9 rate to larotrectinib in pediatric patients with solid ETV6–NTRK3 translocations are recurrent in infan- tumors harboring confirmed NTRK1, NTRK2 or tile fibrosarcoma, reported in approximately 70% of NTRK3 fusions.33 Another report describes treating cases.10 This rearrangement results in a fusion a patient with infantile fibrosarcoma with the novel protein between NTRK3 (also known as NT-3 growth LMNA–NTRK1 fusion with crizotinib, which has factor receptor or TrkC) and the transcription factor been shown to have activity against both NTRK1-and ETV6 with resultant upregulation of the Ras-MAPK NTRK3-dependent tumors.29,34 Both reported and PI3K-Akt pathways.11–15 Molecular techniques patients showed significant responses to these to identify this fusion are a component of the targeted therapies. An additional recent article standard diagnostic work-up for these tumors. describes a patient with secretory breast carcinoma Congenital mesoblastic nephroma is a renal tumor and an ETV6–NTRK3 fusion who also responded to of infancy that, like infantile fibrosarcoma, is targeted TRK inhibition.35 typically diagnosed within the first year of life. In order to treat patients with targeted therapies, Surgical resection is adequate treatment in most however, the presence of the target must be con- cases, with a 5-year survival of ~ 95%.16 Based on firmed, yet the current standard assays (ETV6 break- histologic appearance, congenital mesoblastic apart FISH or ETV6–NTRK3 RT-PCR) will miss cases nephromas are divided into classic, cellular and with alternative fusion partners. In order to establish mixed types, with the cellular type being histologi- whether the EML4–NTRK3 fusion is recurrent, and to cally identical to infantile fibrosarcoma. Like infan- evaluate testing strategies that would capture alter- tile fibrosarcoma, cellular congenital mesoblastic native fusions, we collected archival cases whose nephroma typically harbors the ETV6–NTRK3 differential diagnosis included infantile fibrosar- rearrangement.17,18 The ETV6–NTRK3 fusion has coma, congenital mesoblastic nephroma, mammary also been shown to be oncogenic in multiple analog secretory carcinoma or secretory breast additional tumor types, including mammary analog carcinoma, and screened these cases for rearrange- secretory carcinoma, secretory breast carcinoma and ments using either novel FISH probes, targeted – papillary thyroid carcinoma.19 24 multiplex sequencing, or both. At the time of diagnosis, suspected cases of infantile fibrosarcoma and congenital mesoblastic nephroma are routinely assessed for the ETV6– Materials and methods NTRK3 rearrangement in order to support the histopathologic diagnosis. ETV6 break-apart fluores- Case Selection cence in situ hybridization (FISH) or reverse Sixty-three archival cases from 63 patients of known transcriptase-PCR (RT-PCR) specific to the ETV6– or suspected infantile fibrosarcoma, congenital NTRK3 fusion are the most common tests used to – mesoblastic nephroma, mammary analog secretory screen for this rearrangement.11,21,22,25 27 These carcinoma or secretory breast carcinoma were techniques are necessary as the t(12;15) associated collected from eight institutions. The original sur- with ETV6–NTRK3 fusion is not always visible on geries occurred between 1980 and 2015. Case karyotype, and karyotype may not always be suc- demographics are detailed in Table 1. Note that cessful or available. NTRK3 immunohistochemical one case was previously reported.28 staining is generally not reliable in detecting ETV6– NTRK3 rearranged tumors.14,15 ETV6 break-apart FISH is negative in approxi- Fluorescence In Situ Hybridization mately 30% of infantile fibrosarcoma cases, however, suggesting the possibility of alternate NTRK3 break-apart FISH. Custom FISH probes translocations in this tumor type.13 Recently, case flanking the NTRK3 gene were designed with Agilent reports of novel translocations have been described SureFISH software. The green probe set, comprised in infantile fibrosarcoma, including EML4–NTRK3 of 46 610 oligos spans a region of 400.295 kbp around and LMNA–NTRK1,28,29 whereas cases of ETV6 the 5′ end of NTRK3. The orange/red probe set, fused to unknown gene partners, referred to as comprised of 42 417 oligos, spans a region of 398.66 ETV6-X, have been reported in mammary analog kbp around the 3′ end of NTRK3.

Modern Pathology (2018) 31, 463–473 Table 1 All cases, with patient demographic data, description of tumor, and prior fusion analysis presented on the left

Prior testing Current testing

1 5m M Hand Infantile fibrosarcoma 48, XY, +11, t(12;15) + + Pass ETV6–NTRK3 (p13;q25), +17 [15]/ 49, idem, +20 [1]; 49, idem, +19/ [1]; 46, XY [3] 2 6m F Thigh Infantile fibrosarcoma 46,XX[18]/46,XX,t(1;3) − ++− Pass ETV6–NTRK3 (p36.1;p21)[1]/46,XX, +14,-15, del(18)(p11.2) [1] 3 7m F Retro- Primitive sarcoma 46,XX −−+ + Pass None peritoneum 4 10m F Leg Infantile fibrosarcoma + + Pass ETV6–NTRK3 5 5m F Kidney Congenital mesoblastic Failed + − Fail ETV6–NTRK3 nephroma 6 13d M Kidney Congenital mesoblastic 46,XY −−− Fail nephroma 7 2y M Kidney Cellular neoplasm Failed −− 8 3m M Lower leg Infantile fibrosarcoma 50,XY,+10,+11,t(12;15) +++ (p13;q25)+13,+20[3] /50, idem,-10,+15[2]/51, idem,+15[5]/52,idem, Church AJ EML4 Recurrent +15, +18[4]/46,XY[6] 10 6m M Hand Infantile fibrosarcoma 46,XY[15] − + − Fail

11 3d M Kidney Malignant neoplasm −− − Fail al et 14 7m M Neck Infantile myofibroma −−

15 5m M Kidney Spindle cell sarcoma 50,XX,add(1)(p36.1),+8, +/ − ++ − – +8,+11, add(12)(p13)?t CMN and IFS in NTRK3 (1:12)(p36.1;p13), t (15;16)(q22;q24),+2[3] /51,idem,+19[7] 16 7m F Pelvic soft Infantile fibrosarcoma + + tissue 17 5m F Neck Infantile fibrosarcoma 49-52,XX,+2,+8,+11, t ++ (12;15)(p13;q25),+17, +18, +20[cp19]/46,XX[1] 18 1m F Kidney Congenital mesoblastic 46,XX,t(5;12)(q31;p13) +/ −−−Fail ETV6–NTRK3 nephroma, cellular [7]/ 47,idem,+11[3]/48, oenPathology Modern idem,+11, +16[4]/46,XX [6] 19 4m F Hand Infantile fibrosarcoma 46,XX − ++ − 20 7d M Duodenum Benign myofibroblastic 46,XY −− lesion 21 3m F Forearm Undifferentiated sarcoma 46,XX −−−−Pass None 22 6m F Kidney Congenital mesoblastic 49,X,-X,+2,+8,+11, t + + Pass ETV6–NTRK3

21)3,463 31, (2018) nephroma, cellular (12;15)(p13;q26),+2[2] 25 1m F Buttock Spindle cell sarcoma Failed Failed 27 7d F Kidney −− Fail – 465 473 oenPathology Modern 466

Table 1 (Continued )

21)3,463 31, (2018) Prior testing Current testing

ETV6– EML4– Fusion Tumor NTRK3 by NTRK3 ETV6 NTRK3 NTRK3 Sequencing identified by Study ID Age Sex location Pathologic diagnosis Karyotype results karyotype RT-PCR FISH FISH FISH quality score sequencing – 473 Congenital mesoblastic nephroma 28 2d M Kidney Congenital mesoblastic −− Fail nephroma 32 2d F Kidney Congenital mesoblastic −− Fail EML4 Recurrent nephroma 33 2d M Kidney Congenital mesoblastic + − Fail nephroma, cellular 34 11d F Neck Infantile fibrosarcoma + − Pass ETV6–NTRK3

35 22d M Upper back Infantile fibrosarcoma + − Fail ETV6–NTRK3 – TK nISadCMN and IFS in NTRK3 36 0d F Kidney Congenital mesoblastic 46,XX −−−Fail nephroma, mixed cellularity 38 1d M Shoulder Infantile 45,XY,+5,+6,+7,+12 −− hemangiopericytoma Church AJ 39 16d F Kidney Congenital mesoblastic −− Fail nephroma 40 1d F Forearm Infantile fibrosarcoma + − al et 41 6d M Kidney Congenital mesoblastic 47,XY,+11 − + + Fail EML4–NTRK3 nephroma 42 3d M Kidney Congenital mesoblastic −− Fail nephroma, classic 43 4m M Leg Infantile fibrosarcoma + − Pass ETV6–NTRK3 44 5m M Apex of chest Infantile fibrosarcoma + − Fail 45 11m F Posterior Infantile fibrosarcoma 50,X,-X,add(5)(p15),+8, − + thorax del(10)(p11.2),+11,+14, +17,+20 46 2d M Kidney Congenital mesoblastic 46,XY −− nephroma 47 5d F Kidney Congenital mesoblastic −− Fail nephroma 48 6m M Hand Infantile fibrosarcoma + − Fail ETV6–NTRK3 49 1d F Kidney Congenital mesoblastic 45,XX,t(1;15;12)(q11.2; ++ nephroma, cellular q25;p13),der(1) 50 8m F Mesentery Congenital mesoblastic −− − Pass None nephroma 51 14y F Breast Secretory carcinoma + + − Pass ETV6–NTRK3 52 17y M Parotid gland Acinic cell carcinoma 46,Y,add(X)(p?)[cp2]/46, − ++ − Pass ETV6–NTRK3 XY, add(16)(q?)[cp2]/46, XY[16] 54 2m F Thigh Infantile fibrosarcoma −− 57 72y M Parotid gland Mammary analog secretory + − + − Pass ETV6–NTRK3 carcinoma 58 1y F Lung Spindle cell sarcoma, 52,XY,+del(2)(p21p23), + − + + Pass EML4–NTRK3 metastatic +8,+add (15)(q25)x2, +17,+19[23]/46,XY[1] 59 57y M −− − Pass None Table 1 (Continued )

Prior testing Current testing

Minor Poorly differentiated salivary adenocarcinoma gland 60 83y M Parotid gland Acinic cell carcinoma −− − 61 61y F Parotid gland Acinic cell tumor + + Fail ETV6–NTRK3 62 48y M Parotid gland Acinic cell carcinoma − + − Fail ETV6–NTRK3 63 63y F Breast Invasive carcinoma with −− secretory features 64 51y M Parotid gland Low grade salivary gland −− − neoplasm of uncertain malignant potential 65 26y M Parotid gland Mammary analog secretory + − Pass ETV6–NTRK3 carcinoma 66 50y F Parotid gland Mammary analog secretory + − carcinoma 67 41y F Breast Infiltrating lobular −−− Fail carcinoma 68 8y M Breast Secretory carcinoma 46,XY −−−Pass None 69 60y F Breast Ductal carcinoma in situ + − Pass ETV6–NTRK3

with focal secretory features Church AJ EML4 Recurrent 70 60y F Breast Secretory carcinoma + − Pass ETV6–NTRK3 71 4m F Kidney Congenital mesoblastic + Fail

nephroma, cellular al et 72 9m M Pelvic soft Spindle cell sarcoma, − Pass None

tissue – 73 4m F Kidney Congenital mesoblastic + Pass ETV6–NTRK3 CMN and IFS in NTRK3 nephroma, cellular 74 10m M Forearm Spindle cell sarcoma −− Pass None 75 1y F Retro- Spindle cell sarcoma 59,XX,+1,i(1)(q10),+2, −−Pass None peritoneum +3, del(7)(q34),+8,+8,+9, +11,+12,add(12)(p13)x2, +13,add(13)(p11.2), +16, +19,+20,+21[8]/60,idem, +18[7] 76 5m F Retro- Spindle cell sarcoma Pass ETV6–NTRK3 peritoneum oenPathology Modern 82 4m F Foot Spindle cell sarcoma 45,XX, der(1) add(1) −− Pass RET– (p36.1), t(1;10)(?q32; KHDRBS1 q11.2),10[15]/46,XX[5]

New data are presented on the right, with the results of NTRK3 break-apart FISH, EML4–NTRK3 dual fusion FISH, fusion sequencing quality score and fusion sequencing results. 21)3,463 31, (2018) – 467 473 Recurrent EML4–NTRK3 in IFS and CMN 468 AJ Church et al

Figure 1 Three cases with the EML4–NTRK3 fusion are identified. Case 3, infantile fibrosarcoma: large abdominal mass composed of medium-sized plump oval cells arranged in broad bands separated by thin-walled, ectatic blood vessels. Nuclei are ovoid, mildly pleomorphic with open chromatin and an inconspicuous single nucleolus. Tumor cells undergoing mitoses and apoptosis are frequently observed. Case 58, infantile fibrosarcoma: cellular spindle cell sarcoma characterized by long intersecting fascicles of uniform spindle cells with scant collagenous stroma. Nuclei are elongated with fine chromatin and inconspicuous nucleoli. Mitoses are frequently observed. Case 41, congenital mesoblastic nephroma: cellular spindle cell tumor vaguely arranged in short fascicles. Tumor cells are uniform with round to ovoid to elongated nuclei with open chromatin and inconspicuous nucleoli. Scant collagenous stroma and frequent mitoses are observed. The histology (a, e, i), positive NTRK3 break-apart FISH results (b, f, j), positive EML4–NTRK3 dual fusion FISH results (c, g, k) and fusion sequence reads (d, h) are shown for each case. Note that the sequencing assay for case 41 failed quality control metrics and did not return a result. The case was tested 20 years after specimen collection.

– EML4 NTRK3 dual fusion FISH. Custom FISH NTRK3, PDGFB, PLAG1, ROS1, SS18, STAT6, probes were designed to specifically detect the – TAF15, TCF12, TFE3, TFG, USP6 and YWHAE. All EML4 NTRK3 fusion (Agilent SureFISH). The green purifications during library preparation were per- probe set, comprised of 131 044 oligos spans a region ′ formed with Agencourt AMPure XP (Beckman of 1.363 Mbp around the 5 end of EML4. The orange/ Coulter, cat # A63882). red probe set, comprised of 165 307 oligos spans a ′ RNA quality was assessed in-line with library region of 1.597 Mbp around the 3 end of NTRK3. generation by the Archer PreSeq RNA QC Assay FISH probes were hybridized to metaphase cells (ArcherDX, cat #AK0043-16), provided with the before use in these experiments in order to confirm Universal Reagent Kit, according to kit protocol. hybridization to the correct chromosome. Cycling and data collection was performed on a Formalin-fixed paraffin-embedded tissue slides ™ μ StepOne Real-Time PCR System (ThermoScienti- cut at 5 m thickness were used, prepared according fic, cat # 4376357). The default threshold of 0.078 to standard laboratory practices at their institutions was used for all samples. of origin. Probes were hybridized according to Final libraries were quantified using the KAPA standard protocols using DAKO Histology FISH Library Quantification Kit (KAPA Biosystems, cat # accessory kits. Fifty nuclei were scored using an KK4824) and pooled to equimolar concentration. orange and green dual filter set and individual The intended concentration of pooled libraries was orange and green filters as needed. Images were confirmed with the KAPA Library Quantification Kit. captured using the Applied Imaging CytoVision Imaging System. Interpretation cutoffs were deter- mined independently for each probe set by the beta inverse calculation. Sequencing Libraries were sequenced on an Illumina NextSeq 500 using NextSeq 500 v2 reagents (Illumina, cat # High-Throughput Sequencing for Fusion Detection FC-404-2005) for paired-end, 150 base pair reads and Forty-four cases were tested with a targeted high- dual index reads. Samples were multiplexed such throughput sequencing assay, using an RNA-based that each library was sequenced to at least 2 million anchored multiplex PCR library preparation method paired reads. The flow cell was loaded with 1.8 pM (ArcherDX). Nucleic acids were extracted from denatured library and 20% PhiX Control v3 (Illu- formalin-fixed paraffin-embedded tissue on mina, cat # FC-110-3001). unstained slides using Agencourt FormaPure (Beck- man Coulter, cat # A33341) according to the ’ manufacturer s total nucleic acid purification proto- Data Analysis col. RNA was quantified with Qubit RNA HS Assay Kit (ThermoScientific, cat # Q32852) after purifica- All sequences were recorded in GRCH37 (hg19) tion. Functional RNA quality was assessed in-line coordinates. Data were analyzed by Archer Analysis with library generation. version 4.0. Briefly, adapter sequences were trimmed Libraries were prepared according to Archer from the reads, then PCR duplicates were collapsed FusionPlex Comprehensive Thyroid and Lung Panel using molecular barcodes to identify unique mole- (ArcherDX, cat # SK0070) Protocol Revision C. cules. Split alignments indicated a putative fusion 200 ng RNA—quantified by Qubit—was used as event and a consensus sequence was built from the input for library generation with Archer Universal unique molecules supporting a given fusion event. RNA Reagent Kit v2 (ArcherDX, cat # AK0040-8), The consensus was BLASTed to the reference Archer™ MBC Adapters Set A for Illumina® genome and fusion annotation was guided by (ArcherDX, cat # SA0040) and the Archer Fusion- RefSeq. Finally, a set of filters was applied to the Plex Sarcoma Panel (ArcherDX, cat # AK0032-8). annotated result to reduce false positives and Gene targets include selected exons of: ALK, characterize a fusion event as known or novel. CAMTA1, CCNB3, CIC, EPC, EWSR1, FKHR, FUS, Interpretation of the fusion sequence was made by GLI1, HMGA2, JAZF1, MEAF6, MKL2, NCOA2, a board-certified molecular genetic pathologist.

Modern Pathology (2018) 31, 463–473 Recurrent EML4–NTRK3 in IFS and CMN AJ Church et al 469

Modern Pathology (2018) 31, 463–473 Recurrent EML4–NTRK3 in IFS and CMN 470 AJ Church et al

Results sufficient material were tested to determine whether EML4 is a recurrent alternate translocation partner in – In order to establish whether EML4 NTRK3 fusions tumors known to carry common ETV6–NTRK3 are recurrent in tumor types known to have recurrent fusions. FISH using this probe set was technically – ETV6 NTRK3 fusions, as well as to evaluate testing successful in 45 out of 46 cases. The case previously strategies that would capture alternative fusions, we known to carry the EML4–NTRK3 fusion was conducted a screen of archival cases with a differ- positive using this novel assay, as were two ential diagnosis that included infantile fibrosarcoma, additional cases: a second case of infantile fibrosar- congenital mesoblastic nephroma, mammary analog coma and a case of congenital mesoblastic secretory carcinoma or secretory breast carcinoma. nephroma. The histology in these cases was typical Sixty-three cases of known or suspected infantile of that seen in ETV6–NTRK3 rearranged infantile fibrosarcoma, congenital mesoblastic nephroma, fibrosarcoma and congenital mesoblastic nephroma mammary analog secretory carcinoma and secretory (Figure 1). No cases with a previously detected breast carcinoma were collected from eight institu- ETV6–NTRK3 translocation were positive by tions. Many of these cases had undergone prior this assay. – testing for an ETV6 NTRK3 translocation, typically A subset of the collected cases still did not have an using either ETV6 break-apart FISH or ETV6–NTRK3 identified oncogenic fusion after testing by FISH, RT-PCR. Karyotypes were also available for a subset therefore 44 cases with sufficient tissue were of cases, which may be highly suggestive of an prepared to run on a targeted RNA-based next- ETV6–NTRK3 translocation in the appropriate clin- generation sequencing fusion detection panel ical context (ie, the finding of a t(12;15)(p13;q25) in a (ArcherDX). Twenty-three of these cases passed tumor with histology consistent with IFS). Table 1 pre-sequencing quality control thresholds. The like- provides details of original diagnosis and prior lihood that a case would fail quality control testing. increased with the age of the sample, which is an Prior targeted testing for an ETV6–NTRK3 translo- expected result for an assay using RNA extracted cation had been performed in 23 of 63 (37%) cases. from formalin-fixed paraffin-embedded tissue. Of In addition, 26 (41%) of cases had karyotype testing cases o5 years old at the time of RNA extraction, (without targeted testing). Of the cases with a 95% (19 out of 20) passed quality control. Of the 23 positive finding of ETV6 translocation (with either total cases that passed quality control, 16 fusions FISH or RT-PCR), 100% were given a final patholo- were identified (Table 1), all of which corresponded gic diagnosis of the initially suspected tumor type to the chromosomal alterations identified by FISH in (infantile fibrosarcoma, congenital mesoblastic the current work, or by FISH, karyotype, and/or RT- nephroma, mammary analog secretory carcinoma PCR in previous testing, including 14 cases with or secretory breast carcinoma), whereas of the ETV6–NTRK3 translocation and 2 cases with EML4– negative cases, the final pathologic diagnosis was NTRK3 translocation. In addition, 21 cases did not descriptive (eg, ‘high-grade sarcoma’) in 7 of 13 pass pre-sequencing quality control but were (54%), demonstrating the significance of molecular sequenced anyway. Despite the poor quality scores, findings to the diagnostic process. 7 of the 21 had fusions identified, and 6 of 7 In order to identify all cases of NTRK3 fusion in corresponded to positive FISH results. Five cases this group of cases, regardless of fusion partner, an with poor sequencing quality scores had rearrange- NTRK3 break-apart probe set was created. Interphase ments by FISH that were not identified by sequen- FISH using this probe set was performed on 59 cases cing. On average, these were older cases (7 to 23 and was technically successful in all but one (98%). years, mean 13 years, s.d. 6.4 years from the date of In all, 34 of 58 cases (59%) were positive for an collection to the date of nucleic acid extraction). NTRK3 rearrangement, including 25 of 26 (96%) of In total, of the 44 tumors sequenced, 21 ETV6– infantile fibrosarcoma, 7 of 18 (39%) of congenital NTRK3 fusions were identified, and 2 EML4–NTRK3 mesoblastic nephroma, 3 of 3 (100%) of mammary fusions. Each of the ETV6–NTRK3 fusions identified analog secretory carcinoma and 2 of 3 (67%) of with this analysis connects ETV6 exon 5 secretory breast carcinoma. Eight cases with other (NM_001987.4) to NTRK3 exon 15 diagnoses were negative for NTRK3 rearrangement. (NM_001243101.1). Both of the EML4–NTRK3 All cases that were previously identified to have an fusions identified with this analysis connect EML4 ETV6 rearrangement by either FISH or RT-PCR were exon 2 (NM_019063.4) to NTRK3 exon 14 positive for an NTRK3 rearrangement using the novel (NM_001243101.1). probe set, confirming its ability to detect the Overall, the results of the different targeted assays canonical ETV6–NTRK3 rearrangement. (FISH and/or sequencing) showed good concordance Of the three cases with NTRK3 rearrangement but with each other. For cases tested by NTRK3 break- negative for ETV6 rearrangement by original testing, apart FISH, where another targeted method was also one infantile fibrosarcoma case was known to carry performed, 17 of 19 positive cases, and 12 of 12 an EML4–NTRK3 rearrangement from prior work negative cases correlated with the other result; cases (case 58).28 Therefore, a novel EML4–NTRK3 dual 57 and 62 had conflicting results (Table 1). For cases fusion FISH probe set was created and 46 cases with tested by EML4–NTRK3 dual fusion FISH, where

Modern Pathology (2018) 31, 463–473 Recurrent EML4–NTRK3 in IFS and CMN AJ Church et al 471 another targeted method was available to confirm the result, all results correlated (3 of 3 positive and 34 of 34 negative). For cases tested by sequencing that passed quality control, 15 of 16 positive cases and 7 of 7 negative cases correlated with other results; case 57 had conflicting results, as mentioned above (Table 1). Both of the discordant cases had ETV6 break-apart FISH that was negative at the time of initial work-up, with positive NTRK3 break-apart FISH and ETV6–NTRK3 sequencing results in the current work. Figure 2 The ETV6–NTRK3 and EML4–NTRK3 fusion products are similar, with the 5′ partner contributing the binding domain with conservation of the NTRK3 kinase domain in the 3′ position. Discussion their scope, such that novel fusions such as those Here we describe the recurrent nature of the novel – described here would be missed. EML4 NTRK3 fusion in infantile fibrosarcoma and A multiplex approach to the detection of fusions at congenital mesoblastic nephroma, now detected in 28 the time of diagnosis is recommended for accurate two additional cases beyond the index case. The characterization of tumors whose clinical and histo- fusions include exons 1 and 2 of EML4 and exons 15 logic differential diagnosis includes infantile fibro- through 20 of NTRK3. These breakpoints are similar sarcoma or congenital mesoblastic nephroma. A to those that have been reported in other EML4- and broader approach will also optimize the identifica- – NTRK3 rearranged tumors. The EML4 ALK translo- tion of patients who could potentially benefit from cation is recurrently described in non-small cell lung targeted therapies such as TRK inhibitors. Next- 36 carcinoma. Breakpoints in EML4 are variable in generation sequencing panels that include fusions these tumors, but always preserve the coiled-coil are increasingly available in academic and commer- domain of the EML4 protein (echinoderm cial laboratories. The targeted RNA sequencing assay microtubule-associated protein-like 4), encoded by used here has the advantage of being able to identify 37 exon 2 (NM_019063.4). The chimeric protein fusion partners from a starting point in only one shows constitutive dimerization with resultant acti- partner. For example, by sequencing RNA starting at 36 vation of the tyrosine kinase domain of ALK. The appropriate exon breakpoint regions in NTRK3, one classic infantile fibrosarcoma and congenital meso- can identify ETV6, EML4, or a novel fusion partner. blastic nephroma translocation ETV6–NTRK3 has a Furthermore, the assay can be multiplexed, so that similar functional profile, with preservation of the samples are screened simultaneously for multiple helix loop helix domain of ETV6 and the kinase fusions. For example, a panel could be designed to domain of NTRK3 resulting in oligomerization of the include both NTRK3 fusions and ETV6 fusions, in fusion protein and constitutive activation of the addition to NTRK1, NTRK2, and other significant NTRK3 tyrosine kinase. Although the EML4-NTRK3 soft tissue tumor fusions. A multiplex sequencing translocation is novel, the EML4 and NTRK3 break- approach is particularly helpful when considering a points observed in the current analysis are similar to wide differential diagnosis. A more conservative those that have been previously reported in EML4- approach for pathologists who do not have access to ALK and ETV6–NTRK3 fusions, respectively, and the sequencing panels would be to conduct an initial resulting fusion protein would be predicted to ETV6 break-apart FISH study, which is widely include the coiled-coil domain of EML4 and the available, with a reflex to NTRK3 break-apart FISH kinase domain of NTRK3 (Figure 2). The EML4– if the first result is negative. This approach will NTRK3 fusion has been shown to be tumorigenic require the availability of NTRK3 break-apart FISH. in vitro and in vivo.28 The primary aim of this analysis was to describe Combining these data with previous descriptions the recurrent nature of the EML4–NTRK3 rearrange- of alternate rearrangements in infantile fibrosarcoma ment, thus cases with a range of histologic features and related cancers challenges, the notion that these whose differential diagnosis included infantile fibro- tumors are defined by the ETV6–NTRK3 transloca- sarcoma, congenital mesoblastic nephroma, mam- tion alone. Alternative translocations are increas- mary analog secretory carcinoma or secretory breast ingly described in cancer,38 so it may come as no carcinoma were included. Cases of infantile fibro- surprise that this group of tumors is similarly sarcoma, in particular, which were diagnosed during diverse. Importantly, the diversity and recurrent the era of routine ETV6–NTRK3 testing did not have nature of these novel translocations, as well as their a specific diagnosis made if the fusion was not potential therapeutic significance, requires a reas- detected, which speaks to the importance of mole- sessment of the standard clinical practice of using cular testing in this tumor type. For example, the two ETV6 FISH or ETV6–NTRK3 RT-PCR to identify cases of infantile fibrosarcoma with EML4–NTRK3 fusions. These technologies are robust, but limited in fusions described here were both diagnosed

Modern Pathology (2018) 31, 463–473 Recurrent EML4–NTRK3 in IFS and CMN 472 AJ Church et al

descriptively. Future studies that include compre- NTRK3 oncoprotein binds the phosphotyrosine bind- hensive histopathologic review of large cohorts will ing domain of insulin receptor substrate-1: an essential interaction for transformation. J Biol Chem 2004;279: be needed to describe the frequency of this rearran- – gement in each tumor type and any possible 6225 6234. differences in behavior and outcome. 13 Sheng WQ, Hisaoka M, Okamoto S, et al. Congenital- infantile fibrosarcoma. A clinicopathologic study of 10 Expanding the scope of genetic testing for infantile cases and molecular detection of the ETV6-NTRK3 fibrosarcoma, congenital mesoblastic nephroma, fusion transcripts using paraffin-embedded tissues. Am mammary analog secretory carcinoma and secretory J Clin Pathol 2001;115:348–355. breast carcinoma will result in a better understand- 14 Bourgeois JM, Knezevich SR, Mathers JA, et al. ing of the biology of these tumors and more refined Molecular detection of the ETV6-NTRK3 gene fusion diagnoses for patients. The reliable identification of differentiates congenital fibrosarcoma from other child- gene fusion partners is especially important with the hood spindle cell tumors. Am J Surg Pathol 2000;24: – emergence of targeted therapy, and will allow for 937 946. optimal treatment decisions and improved patient 15 Dubus P, Coindre JM, Groppi A, et al. The detection of outcomes. tel-TrkC chimeric transcripts is more specific than TrkC immunoreactivity for the diagnosis of congenital fibrosarcoma. J Pathol 2001;193:88–94. 16 Malkan AD, Loh A, Bahrami A, et al. An approach to Disclosure/conflict of interest renal masses in pediatrics. Pediatrics 2015;135: 142–158. 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