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Patient with multiple genetically distinct thyroid nodules including papillary thyroid carcinoma harboring novel YWHAG-BRAF fusion

Open AccessPublished:June 24, 2022DOI:https://doi.org/10.1016/j.cancergen.2022.06.006

      Abstract

      Next-generation sequencing (NGS) analysis of thyroid samples aids in risk stratification of cytologically indeterminate nodules and contributes to our understanding of molecular mechanisms in thyroid neoplasia. Several genes, including BRAF, RAS, and EIF1AX, are known to play a role in thyroid tumorigenesis. Here we report a case of papillary thyroid carcinoma (PTC) in which a single lesion harbored a novel YWHAG-BRAF fusion and EIF1AX mutation and displayed mixed morphological findings. The patient is a 74-year-old female with multiple incidentally discovered thyroid nodules, two of which were sampled by ultrasound-guided fine needle aspiration (FNA). Cytologic diagnosis for both nodules was suspicious for follicular neoplasm (Bethesda Category IV). NGS testing of one nodule detected a novel in-frame YWHAG-BRAF fusion and a concurrent EIF1AX A113 splice mutation. The subsequent surgical resection specimen showed that this nodule exhibited two distinct morphologic patterns, conventional (classical) type and follicular variant (FV) of PTC, which were sharply demarcated and were found to harbor unique genetic alterations. Of note, this is the first report of BRAF activation through novel rearrangement with a gene encoding a 14–3–3 protein as a pathogenic factor, which underlines its significance both as a prognostic measurement and as a therapeutic target.

      Keywords

      Introduction

      Up to 30% of biopsied thyroid nodules receive an indeterminate cytologic diagnosis of either “atypia of undetermined significance (AUS)/follicular lesion of undetermined significance (FLUS)” or “follicular neoplasm (FN)/suspicious for follicular neoplasm (SFN)”, Bethesda categories III and IV, respectively [
      • Bongiovanni M.
      • Spitale A.
      • Faquin W.C.
      • Mazzucchelli L.
      • Baloch Z.W.
      The Bethesda system for reporting thyroid cytopathology: a meta-analysis.
      ]. The reported risks of malignancy for these categories are highly variable, complicating the clinical management of these indeterminate nodules. Molecular analysis, notably via next-generation sequencing (NGS), has emerged as an important supplemental tool for evaluating thyroid nodules [
      • Rao S.N.
      • Bernet V.
      Indeterminate thyroid nodules in the era of molecular genomics.
      ]. NGS testing of thyroid fine needle aspiration (FNA) specimens can help with the risk stratification of cytologically indeterminate thyroid nodules and provide insight into the mechanism of thyroid tumorigenesis.
      The most commonly known mutated genes associated with thyroid malignancies are BRAF and RAS. The majority of BRAF mutations are found in classical papillary thyroid carcinoma (PTC), while RAS mutations are seen in follicular variant of papillary thyroid carcinoma (FVPTC) and other malignant and benign follicular-patterned thyroid lesions [
      • Yoo S.K.
      • Lee S.
      • Kim S.J.
      • Jee H.G.
      • Kim B.A.
      • Cho H.
      • et al.
      Comprehensive analysis of the transcriptional and mutational landscape of follicular and papillary thyroid cancers.
      ]. More recently, mutations in other genes such as EIF1AX (Eukaryotic translation initiation factor 1A, X-chromosomal), as well as gene fusions, have been detected in various types of thyroid cancer as well as in some benign thyroid nodules [
      • Simões-Pereira J.
      • Moura M.M.
      • Marques I.J.
      • Rito M.
      • Cabrera R.A.
      • Leite V.
      • et al.
      The role of EIF1AX in thyroid cancer tumourigenesis and progression.
      ,
      • Yakushina V.D.
      • Lerner L.V.
      • Lavrov A.V.
      Gene fusions in thyroid cancer.
      ].
      We report a case of an adult patient with multiple thyroid nodules harboring several distinct genetic abnormalities detected by NGS, which greatly influenced the clinical decision making. One of the molecular alterations is a BRAF fusion with a novel partner gene, YWHAG (also known as 14–3–3 GAMMA or tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein gamma), which has not been previously reported. In addition, a coexisting EIF1AX mutation was identified in that same nodule, which upon surgical resection exhibited two distinct histological patterns of PTC.

      Case presentation

      The patient is a 74-year-old female who presented to the Otolaryngology Clinic for work-up of incidentally discovered thyroid nodules. A brief clinical timeline is shown in Fig. 1. The patient denied any significant symptoms like dysphagia, weight loss, hoarseness or night sweats. She reported a family history of goiter in her aunt and grandmother, but no family history of thyroid cancer. Her physical exam was notable for a normal-sized thyroid with mild nodularity, which moved easily beneath the strap muscles, no palpable cervical lymphadenopathy and normal vocal fold mobility on fiberoptic nasopharyngolaryngoscopy. Ultrasonography of the neck revealed two nodules in the left lobe, one nodule in the left isthmus, two nodules in the right lobe, and no pathologic appearing lymphadenopathy.
      Initially, a 1.2 cm left lobe nodule and the 1.6 cm left isthmic nodule underwent FNA and both received a cytologic diagnosis of “SFN (Bethesda Category IV)”. The FNA specimens were cellular with the follicular cells arranged predominantly in crowded, microfollicular clusters. In addition to this architectural atypia, both nodules also displayed atypical nuclear features such as enlargement and contour irregularities including grooves; therefore, the cytologic differential diagnosis included non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) and FVPTC. Molecular analysis was subsequently performed.
      Total nucleic acids were extracted from selected areas of formalin-fixed, paraffin-embedded (FFPE) tumor tissue with QIAamp DNA FFPE Kit (Qiagen) and from FNA with QIAamp DNA Micro Kit (Qiagen). Archer VariantPlexTM Comprehensive Thyroid and Lung Panel (ArcherDX/Invitae) was used for the detection of gene mutations in genomic DNA. Archer FusionPlexTM Comprehensive Thyroid and Lung Panel (ArcherDX/Invitae) was used for the detection of fusion genes in RNA with the Archer Anchored Multiplex PCR chemistry. This library chemistry allows the detection of both known and unknown fusion partners of queried genes contained in the panel. NGS libraries were sequenced on NextSeq550 Dx (Illumina). Sequencing data were analyzed using the Archer Analysis 6.0 software with bioinformatics pipelines for the detection of gene mutations (SNP/Indel) and gene fusions.
      Molecular testing of the FNA specimen showed a novel YWHAG/BRAF fusion (Fig. 2), along with the EIF1AX A113 splice mutation in the left lobe nodule and the KRAS Q61R mutation in the left isthmus nodule (Table 1).
      Fig 2
      Fig. 2Molecular characterization of FNA sample from 1.2 cm left lobe nodule. NGS revealed an in-frame novel YWHAG-BRAF fusion with 5′ partner (exons 1) of YWHAG and 3′ (exons 11–18) of BRAF. CR2: conserved region 2; RBD: RAS-binding domain.
      Table 1Imaging and laboratory results of all biopsied nodules.
      Nodule Location SizeUltrasound characteristicsFNA CytologyMolecular pathologySurgical pathology
      FusionMutations
      Left lobe

      1.2 cm
      HypoechoicSuspicious for follicular neoplasm (Bethesda IV)Fusion name: YWHAG-BRAF

      Fusion location: Exon–Exon boundary

      Frame status: In-Frame

      5′ partner: End Exon 1 [YWHAG, NM_012479, Breakpoint (RNA): chr7:75,988,039]

      3′ partner: Start Exon 11 [BRAF, NM_004333, Breakpoints (RNA): chr7:140,481,493]
      Gene: EIF1AX

      Position: chrX:20,148,726

      Nucleotide chg.: c.338–1G>C (NM_001412.3)

      AA chg.: A113 splice mutation

      Altered allele freq.: 16.7%
      PTC, mixed conventional type and follicular variant with corresponding molecular findings (see Fig. 3)
      Left isthmus

      1.6 cm
      HypoechoicSuspicious for follicular neoplasm (Bethesda IV)Gene: KRAS

      Position: chr12:25,380,276

      Nucleotide chg.: c.182A>G (NM_004985.4)

      AA chg.: p.Q61R

      Altered allele freq.: 10.5%
      PTC, follicular variant
      Right lobe

      2.2 cm
      Heterogeneous echogenicities including isoechoic, hypoechoic, and

      very hypoechoic
      Suspicious for follicular neoplasm (Bethesda IV)Gene: NRAS

      Position: chr1:115,256,529

      Nucleotide chg.: c.182A>G (NM_002524.4)

      AA chg.: p.Q61R

      Altered allele freq.: 25.4%



      Gene: TP53

      Position: chr17:7,578,403

      Nucleotide chg.: c.527G>A (NM_000546.5)

      AA chg.: p.C176Y

      Altered allele freq.: 2.7%
      PTC, follicular variant
      After cytology and molecular results for the two suspicious left thyroid nodules were reviewed, the recommendation for left thyroid lobectomy and isthmusectomy was made based on the overall risk of malignancy, the size of these nodules, and the absence of more concerning ultrasonographic or physical exam findings. Intraoperatively, she was noted to have several smooth and soft left lobe nodules without gross extrathyroidal extension or central neck lymphadenopathy.
      Surgical pathology examination of the left thyroid lobectomy and isthmusectomy showed two organ-confined foci of PTC. The isthmic nodule showed FVPTC (Table 1). Interestingly, the left lobe nodule exhibited two distinct morphologic patterns, conventional (classical) type and follicular variant (FV) of PTC, with a clear demarcation between them (Fig. 3). Of note, multiple foci of papillary microcarcinoma, a microscopic hyalinizing trabecular tumor, chronic lymphocytic thyroiditis, and nodular thyroid hyperplasia were also identified in the specimen.
      Fig 3
      Fig. 3A-D: The focus of PTC in the left thyroid lobe exhibits two distinct morphologic patterns. The majority of the lesion (A and B) shows follicular architecture (C, higher magnification of the blue inset in B), but an inner well demarcated (black dots in A and B) nodular component contains classical papillary architecture (D, higher magnification of the green inset in B).
      To understand the pathogenesis and the correlation between morphology and molecular findings, we performed molecular analysis on the FFPE tissues from both the conventional type and FV components of the left lobe nodule via microdissection. Interestingly, the conventional PTC component harbored the novel YWHAG-BRAF fusion (35.15% RNA reads) and EIF1AX mutation [c.338–1G>C, altered allele frequency (AAF) 27.21%] concurrently, while the FV component carried only the YWHAG-BRAF fusion (31.9% RNA reads). A minute fraction of EIF1AX mutation (AAF 1.23%) found in the FV component was likely due to contamination during microdissection. Thus, the single left lobe nodule exhibited two distinctive histological variants of PTC, which harbored two different pathogenic gene alterations (i.e., YWHAG-BRAF fusion with and without EIF1AX mutation).
      During the patient's six months post-operative visit, repeat ultrasound showed overall stable nodules in the right lobe including a 2.2 cm upper pole solid hypoechoic nodule. However, given the multifocality of disease in the contralateral thyroid, FNA was recommended for this nodule, which yielded a cytologic diagnosis of “SFN (Bethesda Category IV)”. Molecular pathology revealed two pathogenic mutations, in NRAS and TP53 (Table 1). After a second discussion regarding multifocal thyroid cancer on the left and the risk of malignancy in the right lobe nodule, the patient elected for a completion right thyroidectomy. During that surgery, the surgeon noted a large firm nodule in the right superior pole as well as smaller less firm nodules in the mid/lower pole without evidence of gross extrathyroidal extension, and there was no evidence of lymphadenopathy in the right central neck. Histopathologic examination of the completion thyroidectomy specimen revealed two organ-confined foci of FVPTC. The patient has healed well from surgery with no complications, has had euthyroid labs on 125mcg of levothyroxine daily, and will be followed with thyroglobulin levels.

      Discussion

      Mutation and rearrangement of the BRAF gene play significant roles in tumorigenesis in various cancers, including thyroid cancer, melanoma, gliomas, and lung cancer [
      • Ross J.S.
      • Wang K.
      • Chmielecki J.
      • Gay L.
      • Johnson A.
      • Chudnovsky J.
      • et al.
      The distribution of BRAF gene fusions in solid tumors and response to targeted therapy.
      ]. The activation of B-Raf transduces the signal to the downstream factors and activates the entire Mitogen Activated Protein Kinase (MAPK) pathway. BRAF V600E mutation is the most common (35% to 70%) pathogenic mutation found in PTC [
      • Nikiforov Y.E.
      Molecular analysis of thyroid tumors.
      ,
      • Kimura E.T.
      • Nikiforova M.N.
      • Zhu Z.
      • Knauf J.A.
      • Nikiforov Y.E.
      • Fagin J.A.
      High prevalence of BRAF mutations in thyroid cancer: genetic evidence for constitutive activation of the RET/PTC-RAS-BRAF signaling pathway in papillary thyroid carcinoma.
      ,
      • Xing M.
      BRAF mutation in thyroid cancer.
      ,
      • Cohen Y.
      • Xing M.
      • Mambo E.
      • Guo Z.
      • Wu G.
      • Trink B.
      • et al.
      BRAF mutation in papillary thyroid carcinoma.
      ,
      • He H.
      • Li W.
      • Yan P.
      • Bundschuh R.
      • Killian J.A.
      • Labanowska J.
      • et al.
      Identification of a recurrent LMO7-BRAF fusion in papillary thyroid carcinoma.
      ]. More than 60% of the conventional type of PTC is driven by genetic aberrations involving the BRAF gene [
      • Xing M.
      BRAF mutation in thyroid cancer.
      ,
      • Nikiforova M.N.
      • Kimura E.T.
      • Gandhi M.
      • Biddinger P.W.
      • Knauf J.A.
      • Basolo F.
      • et al.
      BRAF mutations in thyroid tumors are restricted to papillary carcinomas and anaplastic or poorly differentiated carcinomas arising from papillary carcinomas.
      ,
      • Xing M.
      • Westra W.H.
      • Tufano R.P.
      • Cohen Y.
      • Rosenbaum E.
      • Rhoden K.J.
      • et al.
      BRAF mutation predicts a poorer clinical prognosis for papillary thyroid cancer.
      ]. Besides point mutations, BRAF fusion can constitutively activate the MAPK pathway by relieving autoinhibition through the loss of autoinhibitory N-terminal conserved region 1 (CR1) domain of B-Raf [
      • Ciampi R.
      • Knauf J.A.
      • Kerler R.
      • Gandhi M.
      • Zhu Z.
      • Nikiforova M.N.
      • et al.
      Oncogenic AKAP9-BRAF fusion is a novel mechanism of MAPK pathway activation in thyroid cancer.
      ], and plays an important role in tumorigenesis [
      • Stransky N.
      • Cerami E.
      • Schalm S.
      • Kim J.L.
      • Lengauer C.
      The landscape of kinase fusions in cancer.
      ]. More importantly, BRAF V600E mutation or fusions are not found in benign thyroid nodules. To date, at least 20 out of 62 BRAF fusion partner genes (LMO7, AKAP9, SND1, AGK, AP3B1, BCL2L11, FAM11442, MKRN1, MACF1, ZC3HAV1, CCNY, ERC1, SVOPL, OSBPL9, PRKAR2B, SCRIB, KIAA1549, PARP12, CUL1, OPTN) have been identified in thyroid carcinoma [
      • Ross J.S.
      • Wang K.
      • Chmielecki J.
      • Gay L.
      • Johnson A.
      • Chudnovsky J.
      • et al.
      The distribution of BRAF gene fusions in solid tumors and response to targeted therapy.
      ,
      • Hutchinson K.E.
      • Lipson D.
      • Stephens P.J.
      • Otto G.
      • Lehmann B.D.
      • Lyle P.L.
      • et al.
      BRAF fusions define a distinct molecular subset of melanomas with potential sensitivity to MEK inhibition.
      ]. Occasionally, BRAF fusion and V600E mutation can be found concurrently in the same lesion [
      • Stransky N.
      • Cerami E.
      • Schalm S.
      • Kim J.L.
      • Lengauer C.
      The landscape of kinase fusions in cancer.
      ]. In one study, 11 partner genes involved in BRAF fusions were identified in 13 (2.7%) of 484 PTC cases [
      • Agrawal N.
      • Akbani R.
      • Aksoy B.A.
      • Ally A.
      • Arachchi H.
      • Asa S.L.
      • et al.
      Integrated genomic characterization of papillary thyroid carcinoma.
      ]. The majority (8/13) of the BRAF fusion related PTCs show the morphological features of classic variant or tall cell variant PTC, referred to as BRAF V600E-like phenotype [
      • Nikiforov Y.E.
      Molecular analysis of thyroid tumors.
      ,
      • Agrawal N.
      • Akbani R.
      • Aksoy B.A.
      • Ally A.
      • Arachchi H.
      • Asa S.L.
      • et al.
      Integrated genomic characterization of papillary thyroid carcinoma.
      ]. These features are correlated with a significantly poor prognosis [
      • Nikiforova M.N.
      • Kimura E.T.
      • Gandhi M.
      • Biddinger P.W.
      • Knauf J.A.
      • Basolo F.
      • et al.
      BRAF mutations in thyroid tumors are restricted to papillary carcinomas and anaplastic or poorly differentiated carcinomas arising from papillary carcinomas.
      ,
      • Xing M.
      • Westra W.H.
      • Tufano R.P.
      • Cohen Y.
      • Rosenbaum E.
      • Rhoden K.J.
      • et al.
      BRAF mutation predicts a poorer clinical prognosis for papillary thyroid cancer.
      ,
      • Kebebew E.
      • Weng J.
      • Bauer J.
      • Ranvier G.
      • Clark O.H.
      • Duh Q.Y.
      • et al.
      The prevalence and prognostic value of BRAF mutation in thyroid cancer.
      ,
      • Xing M.
      BRAF mutation in papillary thyroid cancer: pathogenic role, molecular bases, and clinical implications.
      ,
      • Namba H.
      • Nakashima M.
      • Hayashi T.
      • Hayashida N.
      • Maeda S.
      • Rogounovitch T.I.
      • et al.
      Clinical implication of hot spot BRAF mutation, V599E, in papillary thyroid cancers.
      ]. In our case, YWHAG-BRAF fusion was found in a nodule showing both classical PTC areas (which also harbored an EIF1AX mutation) and FVPTC areas (which harbored only the BRAF fusion). This is an interesting and unexpected finding, given that BRAF fusions typically occur in classical PTC, while EIF1AX mutations tend to be associated with follicular-patterned thyroid tumors.
      As a discovery, the exact function of the YWHAG-BRAF fusion in tumorigenesis remains unclear. Previous research shows that the BRAF signaling pathway can be activated by losing the autoinhibitory N-terminal CR1 domain, a common consequence in BRAF fusions, including the current YWHAG-BRAF fusion. This mechanism of loss of autoinhibition is different from that of the direct activation of the CR3 kinase domain through phosphorylation induced by BRAF V600E mutation [
      • Ciampi R.
      • Knauf J.A.
      • Kerler R.
      • Gandhi M.
      • Zhu Z.
      • Nikiforova M.N.
      • et al.
      Oncogenic AKAP9-BRAF fusion is a novel mechanism of MAPK pathway activation in thyroid cancer.
      ]. Studies show that altered BRAF can be activated either as a monomer or a dimer [
      • Zaman A.
      • Wu W.
      • Bivona T.G.
      Targeting oncogenic BRAF: past, present, and future.
      ]. Naturally, 14–3–3 proteins can activate BRAF function by direct binding through bridging at the pS729 site of BRAF protein (retained in our fusion protein) and resulting in dimerization [
      • Sun Q.
      • Wang W.
      Structures of BRAF–MEK1–14-3-3 sheds light on drug discovery.
      ,
      • Park E.
      • Rawson S.
      • Li K.
      • Kim B.W.
      • Ficarro S.B.
      • Pino GG del
      • et al.
      Architecture of autoinhibited and active BRAF–MEK1–14-3-3 complexes.
      ]. More interestingly, 14–3–3 gamma isoforms are almost dimeric through the interaction domain, which is partially encoded by exon1 of YWHAG (retained in our YWHAG-BRAF fusion product) [
      • Yang X.
      • Lee W.H.
      • Sobott F.
      • Papagrigoriou E.
      • Robinson C.V.
      • Grossmann J.G.
      • et al.
      Structural basis for protein-protein interactions in the 14-3-3 protein family.
      ]. Thus, BRAF activation can be regulated through two possibilities by the novel YWHAG-BRAF fusion: 1. facilitated dimerization by 14–3–3 gamma encoded by YWHAG; 2. bridging by 14–3–3 gamma. Of note, the breakpoint at exon 11 of the BRAF gene in YWHAG-BRAF fusion is a recurrent site, found in other tumors [
      • Hutchinson K.E.
      • Lipson D.
      • Stephens P.J.
      • Otto G.
      • Lehmann B.D.
      • Lyle P.L.
      • et al.
      BRAF fusions define a distinct molecular subset of melanomas with potential sensitivity to MEK inhibition.
      ,
      • Zaman A.
      • Wu W.
      • Bivona T.G.
      Targeting oncogenic BRAF: past, present, and future.
      ,
      • Lin A.
      • Rodriguez F.J.
      • Karajannis M.A.
      • Williams S.C.
      • Legault G.
      • Zagzag D.
      • et al.
      BRAF alterations in primary glial and glioneuronal neoplasms of the central nervous system with identification of 2 novel KIAA1549: BRAF fusion variants.
      ,
      • Garnett M.J.
      • Marais R.
      Guilty as charged B-RAF is a human oncogene.
      ]. The further study of BRAF fusion and disease prognosis in thyroid carcinoma is warranted.
      Another critical gene found in thyroid cancer tumorigenesis is EIF1AX, which encodes one of the essential factors involved in the initiation of eukaryotic translation. According to one study, EIF1AX mutations are found in 4.2% of thyroid nodules including benign nodules, well-differentiated thyroid carcinomas, poorly differentiated, and anaplastic thyroid cancers [
      • Simões-Pereira J.
      • Moura M.M.
      • Marques I.J.
      • Rito M.
      • Cabrera R.A.
      • Leite V.
      • et al.
      The role of EIF1AX in thyroid cancer tumourigenesis and progression.
      ]. The most common EIF1AX mutations occur in two hotspots: the mutation cluster in exon 2 and the codon A113 splice mutation, which affects the splice sites of c.338–1 G on exon 6 [
      • Simões-Pereira J.
      • Moura M.M.
      • Marques I.J.
      • Rito M.
      • Cabrera R.A.
      • Leite V.
      • et al.
      The role of EIF1AX in thyroid cancer tumourigenesis and progression.
      ,
      • Karunamurthy A.
      • Panebianco F.
      • Hsiao S.J.
      • Vorhauer J.
      • Nikiforova M.N.
      • Chiosea S.
      • et al.
      Prevalence and phenotypic correlations of EIF1AX mutations in thyroid nodules.
      ]. One of these two hotspots, i.e., the codon A113 splice mutation, is identified in our case. The previous data suggest that A113 splice mutation located at exon 6 of EIF1AX is associated with higher grade and aggressive progression in thyroid cancer [
      • Simões-Pereira J.
      • Moura M.M.
      • Marques I.J.
      • Rito M.
      • Cabrera R.A.
      • Leite V.
      • et al.
      The role of EIF1AX in thyroid cancer tumourigenesis and progression.
      ,
      • Agrawal N.
      • Akbani R.
      • Aksoy B.A.
      • Ally A.
      • Arachchi H.
      • Asa S.L.
      • et al.
      Integrated genomic characterization of papillary thyroid carcinoma.
      ,
      • Karunamurthy A.
      • Panebianco F.
      • Hsiao S.J.
      • Vorhauer J.
      • Nikiforova M.N.
      • Chiosea S.
      • et al.
      Prevalence and phenotypic correlations of EIF1AX mutations in thyroid nodules.
      ,
      • Landa I.
      • Ibrahimpasic T.
      • Boucai L.
      • Sinha R.
      • Knauf J.A.
      • Shah R.H.
      • et al.
      Genomic and transcriptomic hallmarks of poorly differentiated and anaplastic thyroid cancers.
      ]. Recent research from our institute reported a 4.5% incidence of EIF1AX mutation in 904 FNA nodules, with 50% mutations occurring at the splice site of c.338–1 G or c.338–2A. Among these nodules, the most common diagnoses are follicular thyroid carcinoma (27%) and follicular variant of papillary thyroid carcinoma (FVPTC, 27%). Notably, the pathological diagnosis correlates with the presence or absence of coexisting mutations with EIF1AX. Overall, an isolated EIF1AX mutation conferred a 36.4% risk of malignancy (ROM), while the ROM was significantly higher in nodules with concurrent RAS, TERT, TP53 mutations (from 71.4% to 100%) [
      • Gargano S.M.
      • Badjatia N.
      • Nikolaus Y.
      • Peiper S.C.
      • Wang Z.-.X.
      Characterization and clinical significance of EIF1AX mutations and co-mutations in cytologically indeterminate thyroid nodules: a 5-year retrospective analysis.
      ], which is consistent with other studies [
      • Agrawal N.
      • Akbani R.
      • Aksoy B.A.
      • Ally A.
      • Arachchi H.
      • Asa S.L.
      • et al.
      Integrated genomic characterization of papillary thyroid carcinoma.
      ,
      • Landa I.
      • Ibrahimpasic T.
      • Boucai L.
      • Sinha R.
      • Knauf J.A.
      • Shah R.H.
      • et al.
      Genomic and transcriptomic hallmarks of poorly differentiated and anaplastic thyroid cancers.
      ].
      It was previously thought that BRAF alteration is mutually exclusive with aberrations in EIF1AX in thyroid cancer, suggesting their distinctive roles in tumorigenesis. However, in our case, the novel YWHAG-BRAF fusion and EIF1AX mutation are found concurrently in the same lesion. The component with conventional PTC morphology harbors YWHAG-BRAF fusion and EIF1AX mutation concurrently, while the follicular variant component carries only the YWHAG-BRAF fusion. As suggested by a previous study, the conventional type of PTC is associated with higher-risk disease and significantly poorer long-term outcomes, including increased risk for disease recurrence and cancer-specific death than the follicular variant of PTC [
      • Henke L.E.
      • Pfeifer J.D.
      • Baranski T.J.
      • Dewees T.
      • Grigsby P.W.
      Long-term outcomes of follicular variant vs classic papillary thyroid carcinoma.
      ]. Our results suggest that the conventional PTC harboring YWHAG-BRAF fusion with concurrent EIF1AX mutation might present a potentially more aggressive malignancy than the FVPTC harboring YWHAG-BRAF fusion only.
      Recently, the highly selective MAP pathway kinases inhibitors, such as Sorafenib and Vemurafenib, have shown significant efficacy for treating tumors harboring BRAF mutations [
      • Zaman A.
      • Wu W.
      • Bivona T.G.
      Targeting oncogenic BRAF: past, present, and future.
      ,
      • Chapman P.B.
      • Hauschild A.
      • Robert C.
      • Haanen J.B.
      • Ascierto P.
      • Larkin J.
      • et al.
      Improved survival with vemurafenib in melanoma with BRAF V600E mutation.
      ]. Increased testing of thyroid cancers, particularly the clinically aggressive forms, for BRAF mutation or rearrangement will facilitate targeted therapy as a more effective and personalized treatment.
      In conclusion, we identified a novel YWHAG-BRAF fusion with a concurrent EIF1AX mutation in a single nodule of PTC with coexisting conventional and follicular variant histology. The results further contribute to understanding the tumorigenesis of thyroid cancer and the insight into the evolution of the tumor driven by different mechanisms, which could eventually improve clinical outcomes by targeted therapy.

      Ethics approval and consent to participate

      Written informed consent of the patient has been obtained.

      Patient consent for publication

      The patient has consented to the submission of the case report to the journal.

      Authors’ contributions

      RL and SG made contributions to writing the manuscript. EC contributed to revising the manuscript, ZW and NB performed the molecular analysis. All authors read and approved the final manuscript.

      Availability of data and materials

      • : The data of the present study are available from the corresponding author upon reasonable request.

      Declaration of Competing Interest

      The authors declare that they have no competing interests.

      Acknowledgements

      Not applicable.

      Funding

      Not applicable.

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