Abstract
Introduction:
Thyroblastoma is a rare and highly aggressive embryonal thyroid malignancy typically associated with DICER1 alterations. However, DICER1-wildtype cases remain poorly characterized at the molecular level.
Methods:
We report a case of aggressive thyroblastoma in a 62-year-old male, negative for canonical DICER1 RNase IIIb mutations. Comprehensive genomic profiling was performed using Oxford Nanopore long-read sequencing, followed by integrative bioinformatic and pathway-level analyses.
Results:
Molecular analysis revealed an alternative oncogenic signature characterized by an EIF1AX p.Lys3_Lys5dup duplication, TERT alterations (promoter C228T and coding p.C42R), and an AGK–BRAF fusion predicted to drive constitutive MAPK/ERK signaling. Functional enrichment analyses highlighted dysregulation of translational initiation, telomere maintenance, and mitogenic pathways, alongside potential immune-escape mechanisms linked to DUX4 activation. Clinically, the tumor exhibited a triphasic morphology, extensive locoregional infiltration, pulmonary metastases, and only transient response to chemotherapy.
Discussion:
These findings expand the molecular spectrum of thyroblastoma beyond the canonical DICER1-driven paradigm and suggest that DICER1-wildtype cases may represent a distinct biological subgroup. The identification of alterations affecting TERT and MAPK pathways highlights potential therapeutic vulnerabilities and supports the clinical value of comprehensive genomic profiling in ultra-rare thyroid malignancies.
Introduction
Thyroblastoma is an embryonal, high-grade thyroid neoplasm recently recognized as a distinct clinicopathological entity and characterized by primitive, multiphenotypic differentiation, aggressive local behavior, and a strong propensity for early dissemination (–). Histologically, thyroblastoma typically displays a heterogeneous architecture that may include epithelial, blastematous, and mesenchymal components, often recapitulating features of fetal thyroid development and ectomesenchymal differentiation (–). Clinically, patients frequently present with bulky cervical disease, airway or mediastinal involvement, and distant metastases at diagnosis, and the prognosis is generally poor despite multimodal management (, , ). Most reported cases harbor recurrent mutations in the DICER1 gene, particularly affecting the RNase IIIb domain, and thyroblastoma has therefore been considered part of the expanding spectrum of DICER1-associated tumors (, , –). In this setting, DICER1 alterations are thought to drive widespread microRNA dysregulation, developmental pathway reactivation, and cellular plasticity, thereby supporting the embryonal phenotype and rapid clinical progression (, –). DICER1 is not considered a driver gene solely because of its mutation frequency, but because multiple lines of genetic and functional evidence support a causal and selectively advantageous role in tumorigenesis. In DICER1-associated neoplasms, mutations show a highly recurrent and non-random pattern, typically involving hotspot missense variants in the RNase IIIb domain that impair 5p microRNA processing while preserving partial protein function. This stereotyped mutational profile, often occurring in a biallelic “two-hit” configuration (germline or somatic loss-of-function plus somatic hotspot mutation), indicates positive selection rather than passenger prevalence. Furthermore, functional studies demonstrate that RNase IIIb mutations lead to global microRNA dysregulation, derepression of oncogenic transcripts, and enhanced cellular proliferation, supporting a direct oncogenic mechanism (). DICER1-wildtype thyroblastomas are exceptionally rare, and their genomic landscape remains essentially undefined (, , ). This represents a major diagnostic and biological gap, because the current diagnostic framework implicitly assumes DICER1 alteration as a hallmark, and consequently, DICER1-intact tumors risk being misclassified as other high-grade thyroid or neck region malignancies (, , ). Parallel to this, advances in comprehensive molecular profiling — including long-read sequencing technologies, structural variant detection, and pathway-level functional annotation — are reshaping our understanding of rare aggressive tumors by uncovering gene fusions, promoter mutations, and non-canonical drivers that are often missed by routine hotspot panels (–). In particular, alterations in EIF1AX, TERT promoter activation, MAPK pathway lesions (including BRAF and BRAF fusion events), and deregulation of transcriptional repressors such as DUX4 have emerged as potentially actionable or prognostically relevant signals in anaplastic, poorly differentiated, and other high-grade thyroid tumors (, , , ). Whether similar mechanisms can substitute for DICER1 loss in thyroblastoma has not been systematically explored.
Here, we report an aggressive thyroblastoma in an adult patient, negative for canonical DICER1 hotspot mutations on targeted analysis, and subsequently characterized through Oxford Nanopore long-read sequencing and integrative bioinformatic and pathway-level analysis. We describe the clinical course, radiologic features, histopathology and immunophenotype, and we define an alternative oncogenic program involving EIF1AX duplication, TERT promoter activation, AGK–BRAF fusion–mediated MAPK/ERK signaling, and DUX4 dysregulation. We also discuss the diagnostic, biological, and therapeutic implications of these findings in the context of DICER1-wildtype thyroblastoma (–, , , –).
Materials and methods
DNA extraction and sequencing
Genomic DNA was isolated from formalin-fixed, paraffin-embedded (FFPE) tumor tissue using the QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany), a silica column–based extraction system optimized for fragmented or cross-linked nucleic acids. DNA concentration and purity were evaluated using a Qubit™ 4 Fluorometer (Thermo Fisher Scientific, USA) and the dsDNA HS Assay Kit, ensuring an A260/A280 ratio of 1.8–2.0 and a minimum yield of 10 ng/µL for downstream analysis. Library preparation was performed following the Oxford Nanopore Technologies (ONT) Ligation Sequencing Kit (Cat. No. SQK-LSK114, formerly 10170100) protocol, which includes DNA repair, end-preparation, and adapter ligation steps. Libraries were loaded onto R10.4.1 flow cells (Cat. No. FLO-MIN114, formerly 10170500) and sequenced on an ONT GridION platform, generating long-read 1D sequences in real time. Basecalling was conducted using Guppy (v6.5) in high-accuracy mode, and output FASTQ files were evaluated for read length distribution and quality metrics using NanoPlot (v1.41.3). Reads with a Phred quality score ≥10 were retained for downstream alignment and variant calling.
Bioinformatic pipeline and variant analysis
Raw Oxford Nanopore sequencing data were basecalled using Guppy (v6.5) in high-accuracy mode to generate quality-filtered reads. Read quality metrics, including read length distribution and Phred score profiles, were assessed using NanoPlot. The resulting FASTQ files were aligned to the human reference genome (GRCh38/hg38) using Minimap2 (v2.26) with parameters optimized for long-read sequencing. Alignment statistics, including mapping rate and coverage distribution, were evaluated using SAMtools.
Small variant calling, including single-nucleotide variants (SNVs) and short insertions/deletions (indels), was performed using Medaka (v1.10), which generates consensus-based variant calls optimized for Oxford Nanopore long-read data. Structural variants (SVs >50 bp) were identified using Sniffles (v2.3) from the long-read alignments.
The initial variant call set was further refined using bcftools by applying quality-control filters based on read depth, mapping quality, and Phred-scaled variant quality score to retain high-confidence calls. Functional annotation of variants was performed using SnpEff (v5.2), which predicted the molecular consequences of each alteration (e.g., missense, nonsense, frameshift, splice-site, and regulatory variants).
Annotated variants were cross-referenced against curated genomic databases, including COSMIC for cancer-associated mutations, ClinVar for clinically interpreted variants, and dbSNP for known polymorphisms. Population allele frequencies from the gnomAD database were used to filter common germline polymorphisms, applying a minor allele frequency threshold of <0.001 in any population.
Because matched germline DNA was not available, variant interpretation relied on a tumor-only filtering strategy. Therefore, reported alterations are described as putative tumor-enriched variants rather than definitively confirmed somatic mutations. To minimize technical artifacts, only variants located on canonical chromosomes (chr1–22, chrX, chrY) were retained, and variants mapped to alternative contigs or low-confidence regions were excluded. Variants were further prioritized based on predicted functional impact, recurrence in cancer databases, and biological plausibility.
Read-level support for candidate variants was manually inspected using Integrative Genomics Viewer (IGV) to confirm consistent strand representation and exclude potential sequencing or alignment artifacts. Structural variant breakpoints and complex rearrangements, including the AGK–BRAF fusion, were additionally visualized using Ribbon. Downstream data processing, statistical summaries, and graphical visualizations were performed using Python (pandas, matplotlib, seaborn) and R (ggplot2).
A limitation of the present study is the absence of matched normal sequencing, which precludes definitive discrimination between somatic and rare germline variants. Nevertheless, the combination of population-frequency filtering, functional annotation, variant prioritization based on cancer-related databases, and manual read-level inspection supports the biological plausibility of the identified high-confidence candidate driver alterations, including the EIF1AX duplication, the TERT promoter hotspot mutation (C228T), and the AGK–BRAF fusion.
Functional enrichment analysis
To investigate the biological significance of the identified genetic alterations, functional enrichment analysis was performed on the list of mutated genes. Analyses were conducted using g: Profiler (v0.3.5) with confirmatory evaluation in Enrichr, querying multiple repositories including KEGG, Reactome, and Gene Ontology (GO) categories (Biological Process, Molecular Function, and Cellular Component). Gene identifiers were standardized to official HGNC symbols prior to analysis. Statistical enrichment was evaluated using a hypergeometric test with Benjamini–Hochberg false discovery rate (FDR) correction for multiple testing. The background gene set consisted of all protein-coding genes annotated in the human reference genome (GRCh38). Pathways and GO terms with adjusted FDR < 0.05 were considered significantly enriched. Enrichment results were exported as tab-delimited files and visualized using R (v4.3.1) with the ggplot2 package. Only genes harboring high-confidence coding or splice-site variants were included in the enrichment analysis.
Statistical analysis
Descriptive statistical analyses were performed to summarize sequencing metrics and variant characteristics. Continuous variables, including variant allele frequency (VAF), read depth, and variant quality scores, were summarized using median and range values. Given the single-case design of the study, no inferential statistical comparisons between groups were performed.
Results
Clinical presentation and radiology
A 62-year-old man with a history of multinodular goiter underwent a total thyroidectomy due to a rapid increase in thyroid size, leading to recent onset of dyspnea and dysphagia. The histological findings were consistent with the diagnosis of thyroblastoma. A postoperative CT scan showed the presence of a cervical mass extending into the anterior mediastinum, infiltrating the right side of the sternal manubrium, right sternocleidomastoid and pectoralis muscles, with multiple metastatic nodes in the Barety lodge and in the aortopulmonary window infiltrating the trachea. Multiple pulmonary metastases were also found. The patient was referred to our center for pathological and molecular consultation. Postoperatively, chemotherapy with Epirubicin and Ifosfamide was initiated; after three cycles, a partial therapeutic response was achieved with dimensional reduction of the lesions. Representative CT scan and PET/CT images (Figures 1a–d) demonstrate the extent of local infiltration and the response after chemotherapy. Treatment was then discontinued due to cardiotoxicity, and the patient was subsequently managed with supportive care only, dying about one month later.
Figure 1
Histopathology and immunophenotype
Macroscopic examination revealed that the right lobe was completely occupied by an 11 cm, gray-yellowish nodular lesion with cystic areas and indistinct margins. A second, smaller nodule measuring 0.7 cm was also identified in the lower third of the left lobe.
Histologically, the neoplasm displayed a heterogeneous, multifocal morphology with three intermixed components. The first component (Figures 2a, b) consisted of primitive microfollicular structures, lacking orderly lobular organization, with irregular connective septa and a rich vascular network. These structures were composed of small cells with hyperchromatic nuclei, containing small lumens with scarce, heterogeneous colloid. In some areas, they were arranged in compact clusters with poorly recognizable luminal spaces. Due to its characteristics, this neoplastic component resembled fetal thyroid tissue.
Figure 2
The second component (Figure 2c) was a solid area of primitive blastema composed of small round cells with scant cytoplasm. These blastematous cells were positive for TTF1, PAX8 (weak), and NSE (weak), but negative for Synaptophysin, SALL4, PLAP, Glypican3, OCT3/4, Inhibin A, and Calretinin.
The third component (Figure 2d) was mesenchymal, consisting of spindle and pleomorphic epithelioid cells, with partial differentiation toward immature osseous and chondroid lineages. These elements were positive for SATB2, CD31, and ERG.
Immunophenotypically, the primitive follicular component showed strong nuclear TTF1 expression (Figure 2e), PAX8 positivity (Figure 2f), and CK AE1/AE3 expression in epithelial cells (Figure 2g). NSE showed weak-to-moderate positivity in the blastematous and microfollicular components (Figure 2h). The tumor exhibited high mitotic activity (22 mitoses/mm²), scattered necrosis, and lymphovascular invasion. All three components were immunohistochemically negative for Calcitonin, S100, p40, CD117, D2-40, cMyc, SSX, and SS18-SSX.
Genetic analysis
Whole-genome sequencing performed on tumor DNA revealed a complex and heterogeneous mutational profile. A total of 2,168 putative somatic variants were initially detected before filtering. Variant allele frequency (VAF) values ranged from 0.01 to 0.98, with a median VAF of approximately 0.46. The distribution displayed a trimodal pattern with peaks corresponding to low-frequency subclonal variants (<0.1), heterozygous events (~0.5), and high-frequency variants approaching clonality (>0.9) (Supplementary Figure 1a). Read depth was predominantly skewed toward low coverage, though a subset of variants reached depths above 1000 reads (Supplementary Figure 1b), potentially indicating focal amplifications or repetitive regions. Similarly, quality scores clustered below 5000, with a small fraction of high-confidence variants exceeding this threshold (Supplementary Figure 1c). The majority of detected variants mapped to autosomal chromosomes, with a notable enrichment on chromosomes 4, 13, and 16. Genomic variants were also unevenly distributed along chromosome coordinates, further reflecting intratumoral genomic instability (Supplementary Figure 1d).
Variant types were mainly single nucleotide polymorphisms (approximately 47%), followed by insertions (~10%) and deletions (~7%) (Figures 3a, b). Following annotation and filtering, high-confidence coding and splice-site variants were selected using SnpEff, and cross-referenced with the COSMIC Cancer Gene Census and ClinVar databases. Among these, pathogenic or likely pathogenic variants were identified in both canonical cancer drivers and poorly characterized genes. Specifically, mutations were found in EIF1AX (p. Lys3_Lys5dup), TERT promoter (C228T), TERT (p.C42R), and a BRAF rearrangement (AGK-BRAF fusion). No pathogenic mutations were detected in the hotspot regions of the DICER1 gene (exons –). Additional variants were observed in less well-characterized loci such as DUX4, OPCML, FAM230C, and ROCK1P1. These alterations indicate the presence of both canonical oncogenic drivers and additional genomic variants.
Figure 3
Functional annotation and GO analysis
The genes harboring pathogenic or likely pathogenic variants were subjected to Gene Ontology (GO) enrichment analysis. Significant enrichment was observed for terms related to translational initiation (linked to EIF1AX), telomere maintenance and cellular senescence (TERT), and MAPK signaling cascade regulation (BRAF) (Figure 3c). Additionally, DUX4 was associated with transcriptional regulation of early embryonic genes and immune-related pathways, supporting a potential role in immune evasion mechanisms. OPCML, a putative tumor suppressor gene, was linked to negative regulation of receptor tyrosine kinase signaling, suggesting a modulating role in oncogenic signaling networks (Figure 3d).
Functional and pathway analysis
An integrated functional and pathway analysis was performed, combining high-confidence variants derived from next-generation sequencing data with externally validated pathogenic alterations. The curated gene list included both rare or poorly characterized genes (e.g., OPCML, DUX4, FAM230C, ROCK1P1, AC008103.3) and clinically relevant drivers (EIF1AX, TERT, BRAF). Gene Ontology (GO) annotation revealed involvement in key biological processes such as translational initiation (EIF1AX), telomere maintenance and cellular senescence regulation (TERT), and signal transduction via the MAPK/ERK cascade (BRAF). Additionally, OPCML was associated with the negative regulation of receptor tyrosine kinases, while DUX4 emerged as a transcription factor capable of activating early embryonic genes and modulating immune-related responses. Pathway enrichment analysis through databases such as KEGG, Reactome, and g: Profiler highlighted several key pathways including protein synthesis regulation, cell cycle control, and telomere elongation. Notably, BRAF was central to the RAS-RAF-MEK-ERK signaling cascade, a canonical pathway involved in cell proliferation and differentiation. The presence of DUX4 suggested the activation of germline and immunomodulatory pathways, consistent with mechanisms of immune escape reported in certain rare tumors. This multi-level enrichment supports the functional relevance of both common and rare genomic alterations in shaping the tumor’s biology. The curated high-confidence variants and their clinical relevance are summarized in Supplementary Table 2.
Clinical impact of molecular alterations
From a clinical and molecular standpoint, the EIF1AX duplication, detected at high variant allele frequency, is consistent with an early clonal event potentially affecting translational regulation. The coexistence of the TERT promoter mutation (C228T) (Supplementary Figure 2a) and the coding variant (C42R) suggests telomerase reactivation, a molecular feature frequently associated with biologically aggressive or poorly differentiated thyroid neoplasms.
The AGK–BRAF rearrangement was supported by multiple split-read alignments spanning the breakpoint region in IGV. A schematic representation of the AGK–BRAF fusion and its downstream MAPK/ERK signaling cascade is provided in Figure 4. Consistent with this observation, the genomic distribution of breakpoint-supporting split reads across the BRAF locus on chromosome 7 demonstrated clustering of split-read signals compatible with the AGK–BRAF structural rearrangement identified by long-read sequencing (Supplementary Figure 2b). DUX4, a transcription factor normally epigenetically silenced in adult tissues, was also identified, further expanding the molecular heterogeneity of this case. In order to explore potential immune-related implications suggested by this finding, additional immunohistochemical analyses were performed. β2-microglobulin showed strong and diffuse membranous expression, whereas HLA class I demonstrated weak and focal staining in tumor cells. CD8 immunostaining revealed a limited and spatially heterogeneous intratumoral cytotoxic T-cell infiltrate, and PD-L1 expression was strong but focal (Supplementary Figure 3). Although these observations do not provide functional evidence of immune escape, they highlight a complex interplay between oncogenic signaling and immune-related pathways, reinforcing the translational relevance of comprehensive molecular profiling in rare thyroid malignancies.
Figure 4
Finally, the presence of alterations in FAM230C and ROCK1P1, whose biological significance remains to be clarified, underscores the heterogeneous and partially atypical genomic landscape characterizing DICER1-wildtype thyroblastoma.
Collectively, these findings illustrate the coexistence of established oncogenic drivers and less-characterized genomic events, reinforcing the molecular complexity of this rare entity.
Discussion
Thyroblastoma represents a recently defined, high-grade embryonal thyroid malignancy characterized by multiphenotypic differentiation and aggressive clinical behavior. Histologically, it typically displays a heterogeneous architecture including epithelial (immature follicular), blastematous, and mesenchymal components, often recapitulating elements of early thyroid development (–). Historically, these tumors were frequently classified under different designations, including malignant thyroid teratoma or carcinosarcoma, before recognition of their distinctive clinicopathological and molecular features (, ).
A defining molecular hallmark of thyroblastoma has been the presence of recurrent mutations in the DICER1 gene, particularly involving hotspot missense variants affecting the RNase IIIb domain (–). These mutations disrupt microRNA processing and lead to widespread dysregulation of gene expression programs, contributing to tumorigenesis through altered developmental signaling pathways (–). Consequently, thyroblastoma has traditionally been considered part of the broader spectrum of DICER1-associated tumors. However, a subset of cases lacks detectable DICER1 mutations, highlighting the existence of alternative oncogenic mechanisms in this rare entity (, , ).
Recent studies have begun to characterize the molecular landscape of DICER1-wildtype thyroblastomas. Xu et al. reported that these tumors frequently lack canonical RNase IIIb mutations and instead harbor alternative oncogenic drivers, including alterations affecting MAPK signaling and telomere-related pathways (). Similarly, Kim et al. demonstrated that aggressive thyroid tumors without DICER1 mutations may rely on canonical oncogenic cascades rather than microRNA-processing defects (). This molecular configuration contrasts with classical DICER1-mutant thyroblastomas, where impaired miRNA biogenesis represents the central pathogenetic mechanism (–). Importantly, it also differs from poorly differentiated thyroid carcinoma, which is typically characterized by mutations in RAS, BRAF V600E, TP53, and frequent TERT promoter alterations arising in a stepwise dedifferentiation model ().
In the present study, long-read genomic sequencing revealed a constellation of alterations involving EIF1AX, TERT, and an AGK–BRAF fusion, defining a potential alternative oncogenic framework. The identification of these alterations expands the molecular spectrum of thyroblastoma and supports the concept that DICER1-wildtype cases may represent a biologically distinct subgroup.
Alterations affecting TERT further reinforce the aggressive biological phenotype observed in this tumor. The promoter mutation C228T generates de novo binding sites for ETS transcription factors and is strongly associated with poor prognosis in thyroid malignancies (35, 36). Because ETS transcription factors are responsive to MAPK signaling, constitutive ERK activation induced by the AGK–BRAF fusion may further amplify TERT transcriptional activity, promoting telomerase activation and replicative immortality (–, 35–37). The coexistence of promoter and coding TERT variants observed in our case suggests a convergent mechanism reinforcing telomere maintenance and cellular immortalization.
The AGK–BRAF fusion represents another key driver identified in this tumor. BRAF fusions are well-recognized oncogenic events capable of activating the MAPK/ERK signaling cascade independently of upstream RAS signaling (–). Although most clinical experience with MAPK-targeted therapy in thyroid cancer has focused on the BRAF V600E mutation, accumulating evidence indicates that BRAF fusion events can similarly drive constitutive pathway activation and may exhibit sensitivity to MEK inhibitors or combined MAPK-targeted therapies (–). The AGK–BRAF rearrangement has been reported in several tumor types, including pediatric papillary thyroid carcinoma and lung adenocarcinoma, and has been associated with enhanced proliferative signaling and tumor progression (, ). The presence of this alteration in thyroblastoma therefore raises the possibility of therapeutically actionable MAPK pathway activation.
To further contextualize this finding, AGK–BRAF fusions have been described as recurrent oncogenic events in pediatric thyroid carcinoma, where they promote constitutive MAPK activation and are associated with distinct biological behavior (38). More broadly, fusion-driven MAPK signaling represents a characteristic feature of pediatric and rare thyroid tumors, often differing from classical BRAF V600E-driven carcinomas in both molecular profile and clinical course (39). In this context, the identification of an AGK–BRAF fusion in thyroblastoma supports the hypothesis that alternative MAPK-activating mechanisms may operate in DICER1-wildtype embryonal thyroid tumors. Notably, according to the WHO Classification of Endocrine and Neuroendocrine Tumours (5th edition), thyroblastoma is currently the only recognized embryonal tumor of the thyroid, underscoring both its biological uniqueness and the lack of representation in large-scale genomic datasets.
The EIF1AX duplication identified in this case likely represents an early clonal event. EIF1AX encodes eIF1A, a translation initiation factor responsible for start-codon recognition and fidelity of the pre-initiation complex. Mutations in EIF1AX have been reported in advanced thyroid carcinomas, where they frequently co-occur with additional oncogenic alterations and contribute to tumor dedifferentiation and aggressive behavior (–32). Mechanistic studies suggest that mutant EIF1AX enhances translation initiation and activates adaptive stress-response pathways involving ATF4, mTORC1 signaling, and c-MYC stabilization, thereby coupling translational control to metabolic reprogramming and proliferation (33, 34). The p. Lys3_Lys5dup variant affects the N-terminal region implicated in scanning dynamics and start-codon selection, potentially altering translational output and contributing to oncogenic signaling.
Functional enrichment analyses further supported the biological relevance of the detected alterations. Gene Ontology and pathway-level analyses highlighted dysregulation of translational initiation, telomere maintenance, and MAPK signaling pathways, consistent with the functional roles of EIF1AX, TERT, and BRAF (–, –37). Additional genes identified in the mutational landscape, including OPCML and DUX4, were associated with modulation of receptor tyrosine kinase signaling and transcriptional programs linked to developmental and immune-related pathways.
The detection of DUX4 is particularly intriguing from an immunological perspective. DUX4 is a transcription factor normally epigenetically silenced in adult somatic tissues but aberrantly expressed in certain malignancies (39–41). Experimental studies have shown that DUX4 activation can suppress interferon-γ–responsive gene programs and impair antigen presentation through downregulation of MHC class I molecules, thereby promoting immune evasion (40–42). In our case, immunohistochemical analysis revealed preserved β2-microglobulin expression, partial reduction of HLA class I, limited CD8-positive T-cell infiltration, and focal PD-L1 expression. Although these findings do not provide definitive functional evidence of immune escape, they are consistent with previously described DUX4-associated immune-modulatory mechanisms. Given the single-case design and the absence of transcriptomic validation, these observations should be interpreted as hypothesis-generating and warrant further investigation through integrated immune profiling approaches.
Taken together, the coexistence of oncogenic alterations affecting translational control (EIF1AX), telomere maintenance (TERT), and MAPK signaling (AGK–BRAF), together with potential immune-modulatory mechanisms associated with DUX4, suggests a composite molecular architecture combining proliferative and immune-related signaling pathways. Although these observations derive from a single case, they may provide a biologically plausible explanation for the aggressive phenotype observed in this tumor and warrant further investigation in additional cases of DICER1-wildtype thyroblastoma.
Overall, our findings demonstrate that thyroblastoma can harbor biologically significant genomic alterations even in the absence of canonical DICER1 mutations. The identification of an alternative molecular framework involving EIF1AX, TERT, and AGK–BRAF expands the current understanding of thyroblastoma pathogenesis and underscores the importance of comprehensive genomic profiling in the diagnostic evaluation of rare thyroid malignancies (–). Future studies involving larger cohorts will be necessary to determine the prevalence and clinical significance of these alterations and to clarify whether DICER1-wildtype thyroblastoma represents a distinct molecular subtype with specific therapeutic vulnerabilities.
Conclusion
Taken together, this report describes an exceptional case of DICER1-wildtype thyroblastoma, a tumor type in which DICER1 alterations are usually considered defining events. The coexistence of EIF1AX, TERT, and AGK–BRAF alterations supports alternative oncogenic mechanisms distinct from canonical DICER1-driven pathways. Functional analysis revealed dysregulation of translation, telomere maintenance, and MAPK signaling, defining a molecularly distinct subset. These findings highlight the importance of integrated histopathological and genomic profiling in clarifying diagnosis and revealing potential therapeutic vulnerabilities in ultra-rare thyroid malignancies.
Statements
Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://zenodo.org/records/17627658,17627658.
Ethics statement
The studies involving humans were approved by IRCCS Istituto Nazionale dei Tumori di Napoli G. Pascale. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.
Author contributions
AG: Writing – original draft, Investigation, Conceptualization. FP: Resources, Writing – original draft, Data curation, Investigation. AD: Validation, Methodology, Data curation, Conceptualization, Writing – original draft, Writing – review & editing. SP: Validation, Methodology, Writing – original draft. ID: Methodology, Writing – original draft. PD: Software, Formal Analysis, Methodology, Writing – original draft. LC: Resources, Writing – original draft, Data curation. AnP: Data curation, Writing – original draft, Resources. AO: Writing – review & editing, Validation, Data curation, Methodology. APe: Writing – original draft, Resources, Methodology. GF: Writing – original draft, Supervision, Writing – review & editing. ST: Writing – original draft, Supervision, Writing – review & editing.
Funding
The author(s) declared that financial support was not received for this work and/or its publication.
Acknowledgments
The authors are grateful to the Ministry of Health for supporting research and to Alessandra Trocino (Librarian at the National Cancer Institute IRCCS Fondazione G. Pascale) for her excellent bibliographic service. We also extend our thanks to the Institutional Biobank-Scientific Directorate of the same Institute for their invaluable experimental support provided during the revision process of this manuscript.
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Generative AI statement
The author(s) declared that generative AI was not used in the creation of this manuscript.
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Supplementary material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fendo.2026.1747919/full#supplementary-material.
References
1
AgaimyANoseVSobrinho-SimõesMWHO Classification of Tumours Editorial Board. Thyroblastoma. In: WHO classification of endocrine and neuroendocrine tumours. International Agency for Research on Cancer, Lyon, France (2022).
2
AgaimyAWitkowskiLStoehrRCuencaJCCGonzález-MullerCABrüttingAet al. Malignant teratoid tumor of the thyroid gland: an aggressive primitive multiphenotypic Malignancy showing organotypical elements and frequent DICER1 alterations—is the term "thyroblastoma" more appropriate? Virchows Arch. (2020) 477:787–98. doi: 10.1007/s00428-020-02853-1. PMID:
3
RooperLM. From Malignant thyroid teratoma to thyroblastoma: evolution of a newly-recognized DICER1-associated Malignancy. Adv Anat Pathol. (2023) 30:136–45. doi: 10.1097/pap.0000000000000364. PMID:
4
ThompsonLDRosaiJHeffessCS. Primary thyroid teratomas: a clinicopathologic study of 30 cases. Cancer. (2000) 88:1149–58. doi: 10.1002/(SICI)1097-0142(20000301)88:5<1149::AID-CNCR27>3.0.CO;2-V
5
CanberkSCorreiaMLimaARBongiovanniMSobrinho-SimõesMSoaresPet al. The multifaceted profile of thyroid disease in the background of DICER1 germline and somatic mutations. J Mol Pathol. (2022) 3:1–14. doi: 10.3390/jmp3010001. PMID:
6
GuilmetteJDias-SantagataDLennerzJSeligMSadowPMHillDAet al. Primary thyroid neoplasm with fetal morphology associated with DICER1 mutations: expanding the diagnostic profile of thyroblastoma. Thyroid. (2022) 32:1423–8. doi: 10.1089/thy.2022.0060. PMID:
7
AgaimyAWitkowskiLStoehrRCuencaJCCGonzález-MullerCABrüttingAet al. Malignant teratoid tumor of the thyroid gland: an aggressive primitive multiphenotypic malignancy showing organotypical elements and frequent DICER1 alterations-is the term "thyroblastoma" more appropriate? Virchows Arch. (2020) 477(6):787–98. doi: 10.1007/s00428-020-02853-1
8
BaiYKakudoKJungCK. Updates in the Pathologic Classification of Thyroid Neoplasms: A Review of the World Health Organization Classification. Endocrinol Metab (Seoul). (2020) 35(4):696–715. doi: 10.3803/EnM.2020.807
9
PillaiSGopalanVSmithRALamAK. Diffuse sclerosing variant of papillary thyroid carcinoma--an update of its clinicopathological features and molecular biology. Crit Rev Oncol Hematol. (2015) 94(1):64–73. doi: 10.1016/j.critrevonc.2014.12.001
10
TarafderKHRahmanSHHossainMAAlauddinMIslamMAHadiIAet al. Outcome of management of differentiated thyroid carcinoma. Mymensingh Med J. (2007) 16(2 Suppl):S46–52.
11
ChenXXiongLLiuHWangHChengDWangWet al. Diagnosis and treatment of thyroblastoma: a case report and review of literature. Front Oncol. (2025) 15:1467631. doi: 10.3389/fonc.2025.1467631
12
ChibaT. Molecular Pathology of Thyroid Tumors: Essential Points to Comprehend Regarding the Latest WHO Classification. Biomedicines. (2024) 12(4):712. doi: 10.3390/biomedicines12040712
13
RooperLMBynumJPMillerKPLinMTGaganJThompsonLDRet al. Recurrent DICER1 hotspot mutations in Malignant thyroid gland teratomas: molecular characterization and proposal for a separate classification. Am J Surg Pathol. (2020) 44:826–33. doi: 10.1097/pas.0000000000001430. PMID:
14
FoulkesWDPriestJRDuchaineTF. DICER1: mutations, microRNAs and mechanisms. Nat Rev Cancer. (2014) 14:662–72. doi: 10.1038/nrc3802. PMID:
15
StewartDRBestAFWilliamsGMHarneyLACarrAGHarrisAKet al. Neoplasm risk among individuals with a pathogenic germline variant in DICER1. Clin Cancer Res. (2019) 25:6198–205. doi: 10.1200/jco.2018.78.4678. PMID:
16
SladeIBacchelliCDaviesHMurrayAAbbaszadehFHanksSet al. DICER1 syndrome: clarifying the diagnosis, clinical features and management implications of a pleiotropic tumor predisposition syndrome. J Clin Oncol. (2011) 29:2312–20. doi: 10.1136/jmg.2010.083790. PMID:
17
HanYLiSZhaoBLvJGaoL. Clinical, molecular and radiological characteristics of thyroid nodules with somatic DICER1 mutations in adults. Endocr Connect. (2025) 14(6):e250125. doi: 10.1530/EC-25-0125
18
Ricarte-FilhoJCCasado-MedranoVReichenbergerESpanglerZScheererMIsazaAet al. DICER1 RNase IIIb domain mutations trigger widespread miRNA dysregulation and MAPK activation in pediatric thyroid cancer. Front Endocrinol (Lausanne). (2023) 14:1083382. doi: 10.3389/fendo.2023.1083382
19
GroisbergRHongDSRoszikJJankuFTsimberidouAMJavleMet al. Clinical Next-Generation Sequencing for Precision Oncology in Rare Cancers. Mol Cancer Ther. (2018) 17(7):1595–601. doi: 10.1158/1535-7163.MCT-17-1107
20
KimYHYoonSJKimMKimHHSongYSJungJWet al. Integrative Multi-omics Analysis Reveals Different Metabolic Phenotypes Based on Molecular Characteristics in Thyroid Cancer. Clin Cancer Res. (2024) 30(4):883–94. doi: 10.1158/1078-0432.CCR-23-2025
21
MechahouguiHFriedlaenderAGörgülüKTsantoulisPIllertALSubbiahVet al. Precision oncology in rare tumors: Have the orphans been adopted? Med. (2026) 7(2):100958. doi: 10.1016/j.medj.2025.100958
22
Di MauroARegaRALeongitoMAlbinoVPalaiaRGualandiAet al. Plexiform fibromyxoma in the stomach: immunohistochemical profile and comprehensive genetic characterization. Int J Mol Sci. (2024) 25:4847. doi: 10.3390/ijms25094847. PMID:
23
XuBGhosseinR. Genomic landscape of poorly differentiated and anaplastic thyroid carcinoma. Endocr Pathol. (2016) 27:205–12. doi: 10.1007/s12022-016-9445-4. PMID:
24
CrispoFNotarangeloT. BRAF inhibitors in thyroid cancer: clinical impact, mechanisms of resistance and future perspectives. Cancers (Basel). (2019) 11:1388. doi: 10.3390/cancers11091388. PMID:
25
SchubertLMarikoML. MAPK pathway inhibitors in thyroid cancer: preclinical and clinical data. Cancers (Basel). (2023) 15:710. doi: 10.3390/cancers15030710. PMID:
26
BottonTYehINelsonTVemulaSSSparattaAGarridoMCet al. Recurrent BRAF kinase fusions in melanocytic tumors offer an opportunity for targeted therapy. Pigment Cell Melanoma Res. (2013) 26(6):845–51. doi: 10.1111/pcmr.12148
27
ChangCCHsiehMSHsuWH. An unusual case of acquired AGK–BRAF gene fusion in metastatic EGFR-mutant lung adenocarcinoma: a case report. J Cancer Res Pract. (2025) 12:20–3. doi: 10.4103/ejcrp.ejcrp-d-24-00023. PMID:
28
CordioliMIMoraesLCarvalheiraGSisdelliLAlvesMTDelceloRet al. AGK–BRAF gene fusion is a recurrent event in sporadic pediatric thyroid carcinoma. Cancer Med. (2016) 5:1535–41. doi: 10.1002/cam4.698. PMID:
29
AlohaliSPayneAEPusztaszeriMRajabMForestVIHierMPet al. Effect of having concurrent mutations on the degree of aggressiveness in patients with thyroid cancer positive for TERT promoter mutations. Cancers (Basel). (2023) 15:413. doi: 10.3390/cancers15020413. PMID: . Published 2023 Jan 8.
30
SakoAMatsuseMSaenkoVTanakaAOtsuboRMoritaMet al. TERT promoter mutations increase tumor aggressiveness by altering TERT mRNA splicing in papillary thyroid carcinoma. J Clin Endocrinol Metab. (2024) 109:e1827–38. doi: 10.1210/clinem/dgae220. PMID:
31
Simões-PereiraJMouraMMLeiteV. The role of EIF1AX in thyroid cancer tumourigenesis and progression. J Endocrinol Invest. (2019) 42:313–8. doi: 10.1007/s40618-018-0919-8. PMID:
32
LandaIIbrahimpasicTBoucaiLSinhaRKnaufJAShahRHet al. Genomic and transcriptomic hallmarks of poorly differentiated and anaplastic thyroid cancers. J Clin Invest. (2016) 126:1052–66. doi: 10.1172/JCI85271. PMID:
33
KrishnamoorthyGPDavidsonNRLeachSDZhaoZLoweSWLeeGet al. EIF1AX and RAS mutations cooperate to drive thyroid tumorigenesis through ATF4 and c-MYC. Cancer Discov. (2019) 9:264–81. doi: 10.1158/2159-8290.CD-18-0606. PMID:
34
Martin-MarcosPZhouFKarunasiriCZhangFDongJNandaJet al. eIF1A residues implicated in cancer stabilize translation preinitiation complexes and favor suboptimal initiation sites in yeast. Elife. (2017) 6:e31250. doi: 10.7554/eLife.31250. PMID: . Published 2017 Dec 5.
35
LiuXQuSLiuRShengCShiXZhuGet al. TERT promoter mutations and their association with BRAF V600E mutation and aggressive clinicopathological characteristics of thyroid cancer. J Clin Endocrinol Metab. (2014) 99:E1130–6. doi: 10.1210/jc.2013-4048. PMID:
36
RenaudERiegelKRomeroRSuryamohanKDistlerUTenzerSet al. Multiomic analysis of papillary thyroid cancers identifies BAIAP2L1-BRAF fusion and requirement of TRIM25, PDE5A and PKCδ for tumorigenesis. Mol Cancer. (2022) 21:195. doi: 10.1186/s12943-022-01665-y. PMID: . Published 2022 Oct 10.
37
SisdelliLCordioliMICVVaismanFMoraesLColozza-GamaGAAlvesPAGet al. AGK-BRAF is associated with distant metastasis and younger age in pediatric papillary thyroid carcinoma. Pediatr Blood Cancer. (2019) 66:e27707. doi: 10.1002/pbc.27707. PMID:
38
CherellaCEWassnerAJ. Pediatric thyroid cancer: recent developments. Best Pract Res Clin Endocrinol Metab. (2023) 37:101715. doi: 10.1016/j.beem.2022.101715. PMID:
39
BalasubramanianAJohnTAsselin-LabatML. Regulation of the antigen presentation machinery in cancer and its implication for immune surveillance. Biochem Soc Trans. (2022) 50(2):825–37. doi: 10.1042/BST20210961
40
ChewGLCampbellAEDe NeefESutliffNAShadleSCTapscottSJet al. DUX4 Suppresses MHC Class I to Promote Cancer Immune Evasion and Resistance to Checkpoint Blockade. Dev Cell. (2019) 50(5):658–671.e7. doi: 10.1016/j.devcel.2019.06.011. PMID:
41
Bello PinedaJMBradleyKR. DUX4 is a common driver of immune evasion and immunotherapy failure in metastatic cancers. eLife. (2024) 12:RP89017. doi: 10.7554/elife.89017. PMID:
42
TaylorBCBalkoJM. Mechanisms of MHC-I downregulation and role in immunotherapy response. Front Immunol. (2022) 13:844866. doi: 10.3389/fimmu.2022.844866. PMID:
Summary
Keywords
AGK–BRAF fusion, DICER1-wildtype, EIF1AX, long-read sequencing, pathway enrichment, precision oncology, TERT promoter mutation, thyroblastoma
Citation
Gualandi A, Picozzi F, Di Mauro A, Pelotti S, D’Arbitrio I, De Luca P, Cannella L, Pizzolorusso A, Ottaiano A, Peddio A, Ferrara G and Tafuto S (2026) Genomic profiling of a DICER1-wildtype thyroblastoma reveals AGK-BRAF fusion, EIF1AX duplication, and TERT promoter mutations: integrated genomic and pathway analysis. Front. Endocrinol. 17:1747919. doi: 10.3389/fendo.2026.1747919
Received
17 November 2025
Revised
19 March 2026
Accepted
31 March 2026
Published
22 April 2026
Volume
17 - 2026
Edited by
Umberto Malapelle, University of Naples Federico II, Italy
Reviewed by
Yanlin Chen, Chongqing Health Center for Women and Children, China
Moein Rajaei, Yale University, United States
Updates
Copyright
© 2026 Gualandi, Picozzi, Di Mauro, Pelotti, D’Arbitrio, De Luca, Cannella, Pizzolorusso, Ottaiano, Peddio, Ferrara and Tafuto.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Annabella Di Mauro, annabella.dimauro@istitutotumori.na.it
†These authors share first authorship
‡These authors share last authorship
Disclaimer
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