Genetic Characterization of Pediatric Sarcomas by Targeted RNA Sequencing
2020; Elsevier BV; Volume: 22; Issue: 10 Linguagem: Inglês
10.1016/j.jmoldx.2020.07.004
ISSN1943-7811
AutoresMatthew R. Avenarius, Cecelia Miller, Michael Arnold, Selene C. Koo, Ryan D. Roberts, Martin Hobby, Thomas W. Grossman, Yvonne Moyer, Richard K. Wilson, Elaine R. Mardis, Julie M. Gastier‐Foster, Ruthann Pfau,
Tópico(s)Protein Degradation and Inhibitors
ResumoSomatic variants, primarily fusion genes and single-nucleotide variants (SNVs) or insertions/deletions (indels), are prevalent among sarcomas. In many cases, accurate diagnosis of these tumors incorporates genetic findings that may also carry prognostic or therapeutic significance. Using the anchored multiplex PCR–based FusionPlex system, a custom RNA sequencing panel was developed that simultaneously detects fusion genes, SNVs, and indels in 112 genes found to be recurrently mutated in solid tumors. Using this assay, a retrospective analysis was conducted to identify somatic variants that may have assisted with classifying a cohort of 90 previously uncharacterized primarily pediatric sarcoma specimens. In total, somatic variants were identified in 45.5% (41/90) of the samples tested, including 22 cases with fusion genes and 19 cases with SNVs or indels. In addition, two of these findings represent novel alterations: a WHSC1L1/NCOA2 fusion and a novel in-frame deletion in the NRAS gene (NM_002524: c.174_176delAGC p.Ala59del). These sequencing results, taken in context with the available clinical data, indicate a potential change in the initial diagnosis, prognosis, or management in 27 of the 90 cases. This study presents a custom RNA sequencing assay that detects fusion genes and SNVs in tandem and has the ability to identify novel fusion partners. These features highlight the advantages associated with utilizing anchored multiplex PCR technology for the rapid and highly sensitive detection of somatic variants. Somatic variants, primarily fusion genes and single-nucleotide variants (SNVs) or insertions/deletions (indels), are prevalent among sarcomas. In many cases, accurate diagnosis of these tumors incorporates genetic findings that may also carry prognostic or therapeutic significance. Using the anchored multiplex PCR–based FusionPlex system, a custom RNA sequencing panel was developed that simultaneously detects fusion genes, SNVs, and indels in 112 genes found to be recurrently mutated in solid tumors. Using this assay, a retrospective analysis was conducted to identify somatic variants that may have assisted with classifying a cohort of 90 previously uncharacterized primarily pediatric sarcoma specimens. In total, somatic variants were identified in 45.5% (41/90) of the samples tested, including 22 cases with fusion genes and 19 cases with SNVs or indels. In addition, two of these findings represent novel alterations: a WHSC1L1/NCOA2 fusion and a novel in-frame deletion in the NRAS gene (NM_002524: c.174_176delAGC p.Ala59del). These sequencing results, taken in context with the available clinical data, indicate a potential change in the initial diagnosis, prognosis, or management in 27 of the 90 cases. This study presents a custom RNA sequencing assay that detects fusion genes and SNVs in tandem and has the ability to identify novel fusion partners. These features highlight the advantages associated with utilizing anchored multiplex PCR technology for the rapid and highly sensitive detection of somatic variants. Sarcomas are a heterogeneous group of neoplasms derived from mesenchymal tissues, with >100 subtypes reported to date.1Fletcher C.D.M. Bridge J.A. Hogendoorn P.C.W. Mertens F. WHO Classification of Tumours of Soft Tissue and Bone. IARC, Lyon, France2013Google Scholar These tumors are broadly categorized as malignancies of the bone or soft tissue and typically manifest in muscle, adipose tissue, or cartilage. Although sarcomas account for approximately 1% of adult solid tumors, epidemiologic studies demonstrate that sarcomas represent approximately 21% of solid tumors in the pediatric population, revealing a clearly disproportionate distribution between the two populations.2Burningham Z. Hashibe M. Spector L. Schiffman J.D. The epidemiology of sarcoma.Clin Sarcoma Res. 2012; 2: 14Crossref PubMed Google Scholar The diagnostic workup of sarcomas routinely relies on integrating patient history and physical examination findings with the characterization of biopsied material and imaging studies. The findings from these studies are evaluated and managed by a multidisciplinary team of experts to establish a diagnosis, evaluate for malignancy, and stage the disease. Ancillary techniques, such as immunohistochemistry, cytogenetics, and molecular genetic testing, may also be incorporated into the workup as adjunctive or confirmatory testing to further assist with the classification of tumors and to guide clinical management.3von Mehren M. Randall R.L. Benjamin R.S. Boles S. Bui M.M. Ganjoo K.N. George S. Gonzalez R.J. Heslin M.J. Kane J.M. Keedy V. Kim E. Koon H. Mayerson J. McCarter M. McGarry S.V. Meyer C. Morris Z.S. O'Donnell R.J. Pappo A.S. Paz I.B. Petersen I.A. Pfeifer J.D. Riedel R.F. Ruo B. Schuetze S. Tap W.D. Wayne J.D. Bergman M.A. Scavone J.L. Soft tissue sarcoma, version 2.2018, NCCN clinical practice guideline in oncology.J Natl Compr Canc Netw. 2018; 16: 536-563Crossref PubMed Scopus (362) Google Scholar,4Schaefer I.M. Cote G.M. Hornick J.L. Contemporary sarcoma diagnosis, genetics, and genomics.J Clin Oncol. 2018; 36: 101-110Crossref PubMed Scopus (62) Google Scholar The genetic characterization of sarcomas was established by early studies that identified recurrent abnormalities, primarily fusion genes, in association with some sarcoma subtypes.5Mertens F. Antonescu C.R. Mitelman F. Gene fusions in soft tissue tumors: recurrent and overlapping pathogenetic themes.Genes Chromosomes Cancer. 2016; 55: 291-310Crossref PubMed Scopus (77) Google Scholar For example, the PAX/FOXO1 fusion is highly specific for alveolar rhabdomyosarcomas, whereas the EWSR1/FLI1 fusion is found in Ewing sarcomas.6Skapek S.X. Ferrari A. Gupta A.A. Lupo P.J. Butler E. Shipley J. Barr F.G. Hawkins D.S. Rhabdomyosarcoma.Nat Rev Dis Primers. 2019; 5: 1Crossref PubMed Scopus (241) Google Scholar,7Grunewald T.G.P. Cidre-Aranaz F. Surdez D. Tomazou E.M. de Alava E. Kovar H. Sorensen P.H. Delattre O. Dirksen U. Ewing sarcoma.Nat Rev Dis Primers. 2018; 4: 5Crossref PubMed Scopus (326) Google Scholar Most commonly, detection of fusion genes is performed by fluorescent in situ hybridization or RT-PCR.8Lazar A. Abruzzo L.V. Pollock R.E. Lee S. Czemiak B. Molecular diagnosis of sarcomas: chromosomal translocations in sarcomas.Arch Pathol Lab Med. 2006; 130: 1199-1207Crossref PubMed Google Scholar These techniques are able to rapidly identify common translocations with a high degree of specificity and sensitivity, which makes them the currently favored method for fusion gene detection.9Bridge J.A. The role of cytogenetics and molecular diagnostics in the diagnosis of soft-tissue tumors.Mod Pathol. 2014; 27: S80-S97Crossref PubMed Scopus (10) Google Scholar However, these techniques are limited in their scalability, they may require knowledge of both fusion partners (RT-PCR and locus-specific fluorescent in situ hybridization), and formalin-fixed, paraffin-embedded material is more challenging to test.10Tanas M.R. Goldblum J.R. Fluorescence in situ hybridization in the diagnosis of soft tissue neoplasms: a review.Adv Anat Pathol. 2009; 16: 383-391Crossref PubMed Scopus (57) Google Scholar Therefore, certain cases may require alternative strategies, including next-generation sequencing–based panel testing. In addition to technical limitations, other genetic factors, including alternative breakpoints, promiscuity with noncanonical fusion partners, or the presence of a less common fusion (eg, FUS/ERG or FUS/FEV in Ewing sarcoma), may also elude standard approaches, such as RT-PCR. In these atypical cases, massively parallel sequencing strategies, such as anchored multiplex PCR, an amplicon-based massively parallel sequencing strategy that enriches for a series of user-specified gene targets to identify fusion genes, single-nucleotide variants (SNVs), and insertions/deletions (indels), have been successfully used to identify a host of recurrent abnormalities in both clinical and research settings (Supplemental Figure S1).11Zheng Z. Liebers M. Zhelyazkova B. Cao Y. Panditi D. Lynch K.D. Chen J. Robinson H.E. Shim H.S. Chmielecki J. Pao W. Engelman J.A. Iafrate A.J. Le L.P. Anchored multiplex PCR for targeted next-generation sequencing.Nat Med. 2014; 20: 1479-1484Crossref PubMed Scopus (590) Google Scholar, 12Lam S.W. Cleton-Jansen A.M. Cleven A.H.G. Ruano D. van Wezel T. Szuhai K. Bovée J.V.M.G. Molecular analysis of gene fusions in bone and soft tissue tumors by anchored multiplex PCR-based targeted next-generation sequencing.J Mol Diagn. 2018; 20: 653-663Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 13Zhu G. Benayed R. Ho C. Mullaney K. Sukhadia P. Rios K. Berry R. Rubin B.P. Nafa K. Wang L. Klimstra D.S. Ladanyi M. Hameed M.R. Diagnosis of known sarcoma fusions and novel fusion partners by targeted RNA sequencing with identification of a recurrent ACTB-FOSB fusion in pseudomyogenic hemangioendothelioma.Mod Pathol. 2019; 32: 609-620Crossref PubMed Scopus (77) Google Scholar, 14Olson N. Rouhi O. Zhang L. Angeles C. Bridge J. Lopez-Terrada D. Royce T. Linos K. A novel case of an aggressive superficial spindle cell sarcoma in an adult resembling fibrosarcomatous dermatofibrosarcoma protuberans and harboring an EML4-NTRK3 fusion.J Cutan Pathol. 2018; 45: 933-939Crossref PubMed Scopus (21) Google Scholar, 15Guseva N.V. Jaber O. Tanas M.R. Stence A.A. Sompallae R. Schade J. Fillman A.N. Miller B.J. Bossler A.D. Ma D. Anchored multiplex PCR for targeted next-generation sequencing reveals recurrent and novel USP6 fusions and upregulation of USP6 expression in aneurysmal bone cyst.Genes Chromosomes Cancer. 2017; 56: 266-277Crossref PubMed Scopus (54) Google Scholar The present study aimed to retrospectively evaluate the clinical utility of a 112-gene custom FusionPlex RNA sequencing panel in sarcoma samples that were negative for the clinically ordered RT-PCR fusion gene testing. When available, clinical data were used in combination with the targeted sequencing results to infer diagnoses and assess whether the genetic results obtained may have had a clinical impact. A cohort of 90 diagnostic samples was selected from a pool of 511 cases originally submitted between 2002 and 2017 for clinical RT-PCR fusion testing. The RT-PCR test menu included sarcoma-related gene fusions with consensus primers detecting common breakpoints for five types of primarily pediatric sarcomas: Ewing sarcoma (EWSR1/FLI1, EWSR1/ERG), rhabdomyosarcoma (PAX3/FOXO1, PAX7/FOXO1), desmoplastic small round cell tumor (EWSR1/WT1), synovial sarcoma (SS18/SSX1, SS18/SSX2), and infantile fibrosarcoma and congenital mesoblastic nephroma (ETV6/NTRK3). Of the 511 samples, the selection criteria considered only samples that tested negative for the clinically ordered RT-PCR fusion gene(s) and had a sufficient amount of residual RNA to permit up to two library preparations plus potential orthogonal RT-PCR confirmation of any newly detected somatic variants. To ensure a breadth of samples, further inclusion criteria were stipulated on the basis of the number of clinically ordered RT-PCR fusion gene(s) ordered (ie, single fusion, a subset of two to four fusions, or the entire sarcoma fusion panel). In total, 90 samples were chosen for targeted sequencing. Clinical information, submitted at the time the sample was originally accessioned for clinical RT-PCR fusion gene testing, was curated. This may have included patient age, sex, RT-PCR fusion testing ordered, indication for study/referring diagnosis, pathologic report, including histologic description, site of resection, and any additional information that may have accompanied these internally and externally referred specimens. Before screening, the samples and the accompanying clinical data were de-identified such that the results from the study could not be traced back to the identity of the patient. This study was approved by the Nationwide Children's Hospital Institutional Review Board as exempt research based on the definition of human subjects research defined by the US Department of Health and Human Services (45 CFR part 46 or 21 CFR part 50). At the time of clinical testing, samples were accessioned into the molecular diagnostic laboratory and RNA was extracted from the tumor specimens (snap frozen or optimal cutting temperature embedding compound embedded). Using 250 ng of the originally extracted RNA, cDNA libraries were generated using the customized FusionPlex Solid Tumor Kit (catalog number dSA09260; Archer, Boulder, CO). Furthermore, the libraries were repaired, adenylated, and ligated with Archer adapters for Illumina (San Diego, CA) sequencing, according to the manufacturer's specifications. Two rounds of PCR were performed using universal and gene-specific primers to generate the targeted enriched libraries for Illumina sequencing (Supplemental Table S1). Libraries were quantified (07960140001; Roche, Basal, Switzerland), pooled at a final concentration of 10 pmol/L with 20% PhiX (catalog number FC-110-3001; Illumina), and denatured with sodium hydroxide. Sequencing was performed on the HiSeq 2500 using on-board clustering (catalog numbers PE-402-4002 and FC-402-4023; Illumina). Sequence data were analyzed using Archer Analysis software version 5.1.3 to process, filter, and identify somatic variants. Fusion-positive samples were orthogonally confirmed by Sanger sequencing. Total RNA (1.5 μg) was used to generate cDNA using the High-Capacity Reverse Transcription Kit (catalog number 4368814; ThermoFisher Scientific, Waltham, MA). To amplify the respective fusions, PCR using gene-specific primers (Table 1) was performed using AmpliTaq Gold DNA Polymerase (catalog number N8080241; ThermoFisher Scientific).Table 1RT-PCR Primer Sequences Used to Validate Fusion Detected by the FusionPlex AssayFusionPrimer sequenceEWSR1/CREB1F: 5'-GTAAAACGACGGCCAGagtcactgcacctccatcct -3′R: 5'-CAGGAAACAGCTATGACaacaactccaggggcaatag-3′COL1A1/USP6F: 5'-GTAAAACGACGGCCAGtgttcagctttgtggacctc-3′R: 5'-CAGGAAACAGCTATGACaacgatcaatgctgctgttg-3′EWSR1/FLI1F: 5'-GTAAAACGACGGCCAGccatggatgaaggaccaga-3′R: 5'-CAGGAAACAGCTATGACgaattgccacagctggatct-3′ETV6/NTRK3F: 5'-GTAAAACGACGGCCAGgggctgaggttgtagcactc-3′R: 5'-CAGGAAACAGCTATGACaagggaagcccatcaacctc-3′TPM3/ALKF: 5'-GTAAAACGACGGCCAGaggtggctcgtaagttggtg-3′R: 5'-CAGGAAACAGCTATGACtcgtcctgttcagagcacac-3′EWSR1/ETV1F: 5'-GTAAAACGACGGCCAGatggcactcagcctgcttat-3′R: 5'-CAGGAAACAGCTATGACcaaaaactgccagagctgaa-3′PDE4DIP/NTRK1F: 5'-GTAAAACGACGGCCAGgtcaccaaatccctttgagc-3′R: 5'-CAGGAAACAGCTATGAgcactcagcaaggaagacct-3′WHSC1L1/NCOA2F: 5'-GTAAAACGACGGCCAGaccagcttccattacgatgc-3′R: 5'-CAGGAAACAGCTATGAgatggcatagtaggccgaga-3′PAX3/NCOA1F: 5'-GTAAAACGACGGCCAGagacctcttaccagcccaca-3′R: 5'-CAGGAAACAGCTATGAcgggtggacagagaagctcat-3′BCOR/CCNB3F: 5'-GTAAAACGACGGCCAGtgcctatagcgatgtgtttga-3′R: 5'-CAGGAAACAGCTATGAcctcctcatgatttgagcact-3′CDC42BPB/BRAFF: 5'-GTAAAACGACGGCCAGatccaacccaaccaacttca-3′R: 5'-CAGGAAACAGCTATGAcctcgagtcccgtctaccaag-3′TFG/METF: 5'-GTAAAACGACGGCCAGcctttcctttgcaattcagtg-3′R: 5'-CAGGAAACAGCTATGAgatgattccctcggtcagaa-3′KIAA1549/BRAFF: 5'-GTAAAACGACGGCCAGaagccccaagtcaaagatcc-3′R: 5'-CAGGAAACAGCTATGAttttcactgccacatcacca-3′BRD4/NUTM1F: 5'-GTAAAACGACGGCCAGctacgccgctactcccttc-3′R: 5'-CAGGAAACAGCTATGAtgagggcagtctgagtaagga-3′CIC/DUX4F: 5'-GTAAAACGACGGCCAGgaggctcctctccctgtacc-3′R: 5'-CAGGAAACAGCTATGAtactcccctgggacgtgggtg-3′EWSR1/KLF15F: 5'-GTAAAACGACGGCCAGcatgagtggccctgataacc-3′R: 5'-CAGGAAACAGCTATGAttcatcttcagagacgggtga-3′FUS/TFCP2F: 5'-GTAAAACGACGGCCAGgtggttacaaccgcagcag-3′R: 5'-CAGGAAACAGCTATGAtgggtttcatcatggagtttca -3′M13 tails are uppercase, and gene-specific sequences are lowercase.F, forward; R, reverse. Open table in a new tab M13 tails are uppercase, and gene-specific sequences are lowercase. F, forward; R, reverse. The PCR cycling initiated with a 94°C denaturation step that lasted for 10 minutes and was followed by eight cycles of touchdown PCR that consisted of a 94°C denaturation step (30 seconds), a 65°C annealing step (1 minute) that stepped down 1°C per cycle ending, and a 72°C extension step (1 minute). The remaining cycles included a 94°C denaturation step (30 seconds), a 58°C annealing step (1 minute), and a 72°C extension step (1 minute). The PCR concluded with a final extension of 72°C for 5 minutes. The PCR products underwent gel electrophoresis, were visualized on the UVP BioDoc-IT Imaging System (VWR, Radnor, PA), and were purified using the QiaQuick PCR Purification kit (catalog number 28105; Qiagen, Hilden, Germany). The purified PCR products were sequenced using the Big Dye Terminator v3.1 Cycle Sequencing Kit (catalog number 4337454; Applied Biosystems, Foster City, CA) and M13F and M13R primers. Following the sequencing reaction, the samples were purified using the Performa DTR V3 Gel Filtration Cartridges (catalog number 42453; Edge Biosystems, San Jose, CA) and were analyzed on an ABI 3130 Genetic Analyzer (Applied Biosystems). An expert panel, consisting of two anatomic pathologists (M.A.A. and S.K.) and a pediatric oncologist (R.R.), reviewed the clinical data before FusionPlex analysis to infer likely diagnoses. Following FusionPlex sequencing, the clinical data were reviewed a second time to derive a proposed post-FusionPlex testing diagnosis. In aggregate, the expert panel of clinicians and pathologists considered the cases before and after FusionPlex testing to determine whether the targeted sequencing may have impacted the clinical management (therapeutic strategy, risk stratification, or diagnostic classification). Targeted RNA sequencing was performed on 90 primarily pediatric sarcoma specimens from a patient cohort consisting of 39 females and 51 males with average age of 7.34 years (range, 9 days to 63 years). On the basis of available clinical information, the range of initial inferred diagnoses included skeletal muscle tumors [rhabdomyosarcoma (n = 39)]; bone tumors [Ewing sarcoma (n = 9), bone tumor not further specified (n = 5)]; fibroblastic tumors [infantile fibrosarcoma (n = 8), myofibroblastic tumor (n = 1), fibroblastic/myofibroblastic tumor not further specified (n = 2), nodular fasciitis (n = 1)]; kidney tumors [congenital mesoblastic nephroma (n = 2), kidney tumor not further specified (n = 1)]; tumors of uncertain differentiation [desmoplastic small round cell tumor (n = 1), myoepithelial carcinoma (n = 1)]; central nervous system tumors [anaplastic medulloblastoma (n = 1)]; endocrine [poorly differentiated neuroblastoma with pleomorphic features (n = 1)]; vascular tumors [atypical infantile hemangioma (n = 1)]; tumors of the nerve [peripheral nerve sheath tumor (n = 1)]; soft tissue tumors with no further specification (n = 4); and unclassified (n = 12) (Supplemental Table S2). Among the patients sequenced, all samples and controls demonstrated high sequence quality metrics (Supplemental Tables S3 and S4). Somatic variants were detected in 41 cases (41/90; 45.5%), including 22 fusion-positive cases and 19 SNV- or indel-positive cases. Two novel findings were identified, a WHSC1L1/NCOA2 fusion identified in case 22 and an indel variant in the NRAS gene (https://www.ncbi.nlm.nih.gov/nuccore, NM_002524: c.174_176delAGC p.Ala59del) in case 33; all other variants have been previously described. The proportion of cases identified with a variant was different depending on whether the case was initially screened for a single fusion, multiple fusions, or all fusions on the RT-PCR panel. Cases initially screened for a single fusion were more likely to yield a FusionPlex result than those screened for multiple or all fusions on the RT-PCR panel (Supplemental Figure S2). These results were further broken down by the specific RT-PCR fusion(s) ordered (Figure 1A). Samples initially tested for the ETV6/NTRK3 fusion found in infantile fibrosarcoma and congenital mesoblastic nephroma were the highest yielding fusion-positive samples (6 cases of 9), whereas those initially tested for the Ewing and alveolar rhabdomyosarcoma fusions yielded the highest number of SNV- or indel-positive cases (9 cases of 27). When parsed by presumed tissue of origin, fibroblastic/myofibroblastic tumors were the highest yielding fusion-positive samples (7/12), whereas rhabdomyosarcomas had the highest number of SNVs or indels (15/39) (Figure 1B). Of note, these 15 SNV- or indel-positive rhabdomyosarcoma cases were not concurrently found to be fusion positive. Of the 22 fusions detected in this study, 21 have been previously associated with histologic subclassifications of sarcoma. The most frequently observed fusion partner was EWSR1 (n = 4); other fusion partners, including NCOA2 (n = 3), CIC (n = 2), NCOA1 (n = 2), BCOR (n = 2), BRAF (n = 2), and NTRK (n = 2) genes, were also recurrently identified (Figure 2, Supplemental Table S5). To complement the fusion gene analysis, the data set was analyzed for sequence variants in a subset of cancer genes and their known hot spots. A total of 21 SNVs or indels were detected among 19 cases. Alterations were most frequently seen in the RAS family members, TP53, and CTNNB1 (Figure 2, Supplemental Table S6). Of particular interest, a novel in-frame deletion of an alanine residue at amino acid position 59, located in the nucleotide-binding domain, of the NRAS gene was identified in sample 33. Finally, findings in three other genes, JAK3 (p.V722I), RAF1 (p.S257L), and TP53 (p.Cys135Phe, p.Arg273His, p.His179Arg, and p.Pro301GlnfsTer44), may represent germ-line alterations; however, without paired-normal specimens, the etiologic nature of these variants cannot be fully discerned. The apparent impact this additional testing may have had on clinical outcomes was evaluated by considering the FusionPlex variant identified in conjunction with the available clinical information. In 25 of the 90 cases (27.8%), a possible impact to clinical management was noted and included changes in chemotherapy regimen, favoring surgical resection over chemotherapy, or integration of a targeted therapy. In 17 of the 90 cases (18.9%), there was a potential change in diagnosis (ie, rhabdomyosarcoma to Ewing sarcoma) or refinement of the diagnosis (ie, Ewing sarcoma to Ewing-like sarcoma). Finally, the expected prognosis in 17 of the 90 cases (18.9%) may have been impacted by these results (Supplemental Tables S5 and S6). In this study, a custom FusionPlex panel with probes targeting 112 genes previously associated with solid tumorigenesis was designed for the simultaneous detection of recurrent fusion genes, SNVs, and indels. This panel was used to perform retrospective sequencing on a cohort of 90 banked diagnostic sarcoma specimens that were negative for the clinically ordered RT-PCR fusion gene testing. Among this cohort, 22 fusion-positive cases and 19 SNV- or indel-positive cases were identified. As highlighted by this study, pediatric sarcomas negative for first-tier fusion gene testing may benefit from reflex testing to methods such as this custom FusionPlex panel. This study demonstrates the advantages of targeted sequencing using the anchored multiplex PCR technology; however, the retrospective nature of this study has several limitations, including the following: i) incomplete clinical and laboratory (ie, fluorescent in situ hybridization, cytogenetics, and immunohistochemistry) data for some samples, which often made inferring the diagnosis or interpreting the clinical significance of the variants challenging; ii) lack of access to the original specimens, which precluded histopathologic review; iii) several fusions could not be confirmed by RT-PCR, which may in part be because of RNA degradation, low tumor percentage, primer placement, or sequence homology; and iv) absent paired-normal specimens made it challenging to determine the origin, germ line or somatic, for some variants. 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Germ-line transmission of a mutated p53 gene in a cancer-prone family with Li-Fraumeni syndrome.Nature. 1990; 348: 747-749Crossref PubMed Scopus (1034) Google Scholar Despite these limitations, the current results highlight several advantages associated with the FusionPlex system. This includes enhanced sensitivity, which, in part, is achieved by deep sequencing and the utilization of multiple primers to target alternative breakpoints of variant transcripts. This was demonstrated by the detection of an EWSR1/FLI1 fusion in case 1 (Ewing sarcoma) and an ETV6/NTRK3 fusion in case 14 (infantile fibrosarcoma) by the FusionPlex assay. These cases were initially negative for their respective fusions by clinical RT-PCR analysis. Subsequent RT-PCR confirmation required increasing the amount of RNA in the reverse transcription reaction and designing primers that were closer to the breakpoints. It is possible that the initial clinical RT-PCR assay may have been unsuccessful in detecting these fusions because of a fusion-positive tumor fraction that fell below the limit of detection or because of a single-nucleotide polymorphism in a primer binding site. In either case, the increased sensitivity of the FusionPlex panel successfully identified the EWSR1/FLI1 and ETV6/NTRK3 fusions in these cases, whereas initial clinical RT-PCR testing did not. Another advantage of the FusionPlex system is that it can detect challenging fusions, such as CIC/DUX4, which have variant breakpoints that are mediated by homologous DUX and DUX-like gene clusters at the terminal end of 4q and 10q.26Loke B.N. Lee V.K.M. Sudhanshi J. Wong M.K. Kuick C.H. Puhaindran M. Chang K.T.E. 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