Embryonal tumor with multilayered rosettes (ETMR) is a rare tumor type found in young children's central nervous system (CNS). Despite its infrequency, the grim prognosis associated with ETMR has drawn attention from both neuropathologists and oncologists, emphasizing the need for improved diagnostic and therapeutic approaches for this condition. While there is an immunohistochemical test available, such as LIN28A, it is not exclusive to ETMR. ETMR has less than a decade of diagnostic experience, which contributes to its challenging diagnosis and subclassification [1]. This tumor comprises a conglomerate of three morphologically distinct tumor types; embryonal tumor with abundant neuropil and true rosettes ependymoblastoma (ETANTR), ependymoblastoma (EBL), and medulloepithelioma (MEPL) [2]. The underlying molecular biology of ETMR primarily involves the TTYH1:C19MC fusion and amplification of the C19MC oncogenic miRNA cluster, with the second most common driver being a hereditary mutation in the DICER1 gene [3]. Next-generation sequencing (NGS) has proven valuable in diagnosing ETMR, and methylation clustering is also proving to be helpful, particularly in distinguishing ETMR from other primary CNS embryonal tumors [3, 4]. This article presents four different cases of ETMR that have been sub-classified using methylation clustering. These cases exhibit typical characteristics of ETMR, except for one adult case with DICER1 mutation that demonstrates a unique clinicopathological presentation. Additional research endeavors may provide further understanding regarding the prevalence, biological behavior and therapeutic strategy associated with ETMR.

Clinicopathologic assessment

After Institutional Review Board (IRB) approval, a clinical database search was undertaken to identify all diagnosed ETMR between 2016 and 2023 at Seoul National University Hospital (SNUH). Clinical and follow-up information obtained from institutional databases or electronic medical records included age, gender, radiologic assessments, types of surgery, medical management, and vital status at follow-up. At the time of diagnosis, LIN28A IHC was used as a screening tool when the morphology of the tumor suggested ETMR.

Molecular analyses with next-generation sequencing

The molecular genetics integrated diagnosis followed using a next-generation sequencing (NGS) panel called “FiRST Brain Tumor Panel (BTP)”, customized by SNUH as described in Lee et al [5]. The panel includes 14 essential genes and 210 selected genes for DNA samples, as well as 151 genes for RNA samples. Whole genome sequencing results were analyzed for chromosomal structural variations (SVs). Arriba was used to detect and visualize gene fusions from RNA sequencing data.

Methylation array analysis and t-SNE clustering with the Illumina 850K microarray

DNA methylation array analysis was performed for three cases of ETMR using an Infinium MethylationEPIC 850K BeadChip array platform (Illumina, USA). DNA methylation data analysis was performed using R software (R 4.2.0, https://www.r-project.org/). Our samples were analyzed using the methylation classifier of DKFZ with GSE90496, GSE122038 (2,979 samples from 92 subclasses) [3, 6]. A preprocessing procedure was conducted for raw methylation intensity signals using the R package “methylationArrayAnalysis” (version 1.22.0) [7]. We utilized the “combinedArray” command to merge the different platforms (850K, EPIC). After filtering and normalization, 265,398 probes remained for subsequent analysis. The 10,000 most variably methylated probes were selected based on the standard deviation to perform unsupervised nonlinear dimension reduction. The resulting distance matrix was used as the input for t-distributed stochastic neighbor embedding analysis (t-SNE; Rtsne package version 0.15). The non-default parameters used were distance=TRUE, perplexity=30, θ=0, and max_iter=2000. A t-SNE plot was generated using the “ggplot2” package (version 3.4.2) for effective visualization. IDAT files were uploaded to versions v11b4 and v12.5 of the online CNS tumor methylation classifier (https://www.molecularneuropathology.org) for classification.

Ethics approval and consent to participate

This study (IRB No: 1905-108-1035) conducted at Seoul National University Hospital (SNUH) has obtained approval from the institutional review board (IRB) and has been carried out in accordance with the ethical principles outlined in the 1964 Declaration of Helsinki and its subsequent revisions. Since this study involves a retrospective review of de-identified electronic medical records, pathology data, and next-generation sequencing (NGS) data using a brain tumor-specific somatic gene panel, the IRB has waived the requirement for informed consent, in compliance with the Korean Bioethics and Safety Act.

Clinical manifestations

Patient 1 was a previously healthy 4-month-old female who presented with a chief complaint of non-projectile vomiting twice daily for a 2 weeks. Although initially suspected to be acute gastroenteritis, the patient was later admitted when she started experiencing frequent upper eyeball deviation, loss of consciousness, and worsening vomiting. Brain MRI revealed a severe obstructive hydrocephalus, caused by a tumor measuring 4.8 cm in diameter in the 4^th ventricle (Fig. 1A). The patient underwent a midline suboccipital craniotomy to remove the tumor.

Patient 2 was a 6-month-old male infant who suddenly developed an inability to hold his head up, accompanied by an enlargement of the head size and projectile vomiting. Brain MRI revealed a mass measuring 6.1×3.7×3.1 cm in the midline posterior fossa of the brain, resulting in obstructive hydrocephalus (Fig. 1B). The patient underwent midline suboccipital craniotomy and tumor removal.

Patient 3 was a 2-year-old female who experienced episodes of waking up with stiffness and shaking in her body. These symptoms worsened over time, occurring during the daytime as well and affecting her sleep and appetite. A brain MRI was performed after the patient exhibited fewer facial wrinkles on the right side when frowning, revealing a mass measuring 3.0×2.4×2.4 cm in the pineal region (Fig. 1C). The patient underwent a craniotomy and tumor removal. Subsequently, the two times more craniotomies were performed and subtotal resection of the recurring tumors in the pineal gland and posterior fossa, respectively, seven months after the initial operation.

Patient 4 was a 30-year-old male who presented with facial palsy, hearing loss, and tinnitus on the right ear that progressively worsened over the course of a month. Brain MRI revealed a well-enhancing nodular mass measuring 2.3 cm at the cerebellopontine angle, initially suspected to be a vestibular schwannoma. The patient initially received a gamma knife surgery, but four months later, he experienced dizziness, nausea, and vomiting. MRI showed a decrease in the size of the mass to 1.4 cm, leading to conservative care. However, four months later, the patient arrived at the emergency room with a sudden onset of severe headache. MRI revealed an increase in the mass size to 4.3×2.5×2.4 cm (Fig. 1D), prompting the patient to undergo a surgical procedure called retromastoid suboccipital craniotomy for tumor removal.

The clinical and pathological characteristics of these four patients are summarized in Table 1 and the initial location and size of ETMR for each patient during the initial imaging study are depicted in Fig. 1.

Patients 1 to 3 were diagnosed with ETMR and received additional chemotherapy using a combination of drugs [Korean society of pediatric neuro-oncology regimen (KSPNO-S-1102A) (Irinotecan, Cisplatin [CDDP], cyclophosphamide [CPM], vincristine [VCR], etoposide [VP16]), along with peripheral blood stem cell mobilization (PBSCM). Case 4 underwent radiotherapy to the right cerebellopontine angle (30 Gy) and one cycle of combination chemotherapy with vincristine, cyclophosphamide and prednisolone. In all four patients, the latest MRI scans revealed the presence of multiple tumor deposits along the leptomeninges, with the hydrocephalus and unfortunately, expired within 1 year. Patient 1 had the shortest follow-up duration of 3.4 months, while patient 3 had the longest at 13.7 months.

The result of the histopathological and immunohistochemical (IHC) study

Histopathologically, the tumor from patients 1 through 3 exhibited abundant neutrophils with multilayered rosettes, which is consistent with the characteristic features of this type of tumor (Fig. 2). Patient 4 also displayed these features, but additionally showed chondroid differentiation, which aligns with other known tumors associated with DICER1 mutations. The tumors in all cases exhibited a high mitotic rates, with counts ranging from 45 to 145 per 10 high power fields across patient 1 to 4.

Immunohistochemical analysis revealed that all cases exhibited positive staining for LIN28A. Additionally, focal positive staining was observed for synaptophysin, neurofilaments (NF), and glial fibrillary acidic protein (GFAP) (Fig. 2). The Ki-67 labeling indices, which reflect the proliferation rate of the tumor cells, were found to be high in all cases, with indices ranging from 52.4% to 89.4% (mean: 70.5%).

The result of molecular genetic studies

Through NGS tests, it was revealed that patients 1 to 3 exhibited a TTYH1::C19MC fusion along with C19MC amplifications (Fig. 3). However, patient 4 was found to harbor a mutation in the DICER1 gene.

The Arriba plots generated from RNA sequencing analysis provided insights into the genomic alterations of patients 1, 2, and 3. Interestingly, patients 1 and 2, who had the TTYH1::C19MC fusion and C19MC amplification, exhibited chromothripsis as the underlying mechanism, while patient 3 displayed a deletion as the cause of the genomic alteration (Fig. 3). To further elucidate the relationship between these patients, t-SNE clustering analysis was performed. The results demonstrated that patients 1 and 2, with the TTYH1::C19MC fusion and C19MC amplification, clustered together with the C19MC-altered cluster. On the other hand, patient 4, who had a mutation in the DICER1 gene, clustered with the C19MC normal cluster.

ETMRs were first identified as a distinct tumor type in the early 2000s, when Li et al. [8] reported a frequent amplification of the 19q13.41 microRNA amplifications in brain primitive neuroectoderma tumors (PNETs). In 2007, the WHO classified the CNS PNETs subdivided into CNS neuroblastoma/ganglioneuroblastoma, medulloepithelioma (MEPL), ependymoblastoma (EBL), with embryonal tumor with abundant neuropil and true rosettes (ETANTR) discussed as a unique variant [2]. In a landmark paper by Korshunov et al. [9] demonstrated through genome-wide DNA methylation profile analysis of 41 tumors that ENTANTR, MEPL, EBL, and ETANTR, share common LIN28A immunohistochemical positivity and amplification of the chromosome 19q13.42 locus indicating their classification as a single clinicopathological tumor type [2]. Consequently, LIN28A was suggested to screening tool for ETMR [9]. In 2016, the WHO CNS Tumor Classification officially recognized ETMR as a separate tumor entity [10]. However, it was later discovered that LIN28A positivity is not exclusive to ETMR, as it can also be positive in atypical teratoid rhabdoid tumor (ATRT) [1]. Therefore, C19MC amplification testing was also recommended as an additional method to classify a PNET as ETMR [10, 11]. A key study by Lambo et al. [3] revealed that while C19MC amplification is observed in 90% of ETMR cases, the next most common is DICER1 germline mutation, which conserved genomic instabilities due to R-loop structures as identified through whole genome sequencing. In the same year, Uro-Coste et al. [4] reported two cases of ETMR with DICER1 mutations and illustrated them through methylation clustering. However, Lambo et al. [3] pointed out that methylation data alone is insufficient to classify ETMR beyond being C19MC-altered or non-C19MC altered. More recent studies by von Hoff et al. [12] demonstrated 58 ETMR cases out of a cohort of 307 well-characterized and annotated DNA methylation array.

We have conducted a study following the established methods to confirm the LIN28A IHC positivity in all 4 of our patients with ETMR. Moreover, we molecularly verified the presence of C19MC amplification and TTYH1::C19MC fusion, as well as a DICER1 mutation using RNA sequencing, which is a notable advancement considering previous reports primarily relied on DNA NGS. Additionally, Arriba plots generated from the RNA sequencing data provide insights into the amplification site for patients 1, 2, and 3. Furthermore, through comprehensive whole genome sequencing, we identified chromothripsis as the mechanism of fusion for patients 1 and 2, which corroborated the findings previously reported by Lambo et al. [13]. In contrast, the patient 3 exhibited a deletion mechanism. Notably, through methylation clustering analysis, patient 4 was shown to lack C19MC amplification, in accordance with the presence of DICER1 mutation. Our case series to demonstrate the robustness of the diagnostic criteria of the current WHO Classification of Tumors for CNS, emphasizing the importance of considering ETMR morphology, immunohistochemistry, and genetic alteration as essential criteria. Furthermore, when confronted with challenging cases, a DNA methylation profile that aligns with ETMR can provide valuable insight.

The prognosis for ETMR is dismal, primarily due to its epidemiology and rarity. Since the majority of cases occur in the children below the age of 2, with very few cases reported in older children, the use of high-dose chemotherapy is limited by its associated toxicities [13, 14]. Radiation therapy can be effective but it accompanied by several severe side effects [14]. Proton therapy has been suggested as an alternative to reduce side effects compared to conventional radiation therapy [15]. However, targeted therapies may offer a promising approach to minimize side effects. Targeting R-loops with topoisomerase and PARP inhibitors has been proposed as a potential strategy [13]. DNMT3B, which is active only during the early first weeks of neural tube development, has been suggested as a candidate for future therapies even prior to the inclusion of ETMR in the current WHO Classification of Tumors [16]. Furthermore, pathways such as MIR17HG, WNT SHH, or mTOR pathways could be potential targets downstream from the known C19MC amplification and DICER1 germline mutation [13]. Independent prognostic factors that have been reported include complete resection, non-metastatic, and supratentorial locations [14]. However, in our patient cohort, none of patients underwent complete resection, all eventually developed leptomeningeal metastatic spread, and contrary to the unusual occurrence of posterior fossa lesions, the patient with the longest survival (patient 3) had a supratentorial lesion in the pineal gland. Due to the rarity of ETMR and absence of established treatment regimens, early detection and management remain crucial in combating this aggressive tumors.

Patient 4, an adult male aged 30, presented a unique case of ETMR with a DICER1 mutation at our institution, which is an unusual finding given that ETMRs predominantly affect infants. To date, the oldest reported patient with ETMR is 10 years old [13]. The remarkable exception highlights the importance of exploring the biological distinctions between DICER1-mutant ETMR and the more common subtype characterized by C19MC amplification and TTYH1::C19MC fusion. Microscopic examination of the ETMR in patient 4 revealed distinctive features not observed in the ETMRs of the other three patients with C19MC amplification. Specifically, patient 4 exhibited atypical differentiation with the presence of chondroid area that deviated from the recognized morphologic subtype of ETMR. However, it is noteworthy that chondroid differentiation is a recognized feature in the context of DICER1 syndrome-related tumor.

Genetic counseling is strongly recommended for patients with DICER1-mutant ETMR, as the majority of DICER1 mutations associated with cancer are known to have a germline mutation along with a second-hit somatic mutation [10]. The family of patient 4 was also provided with genetic counseling in line with the recommendation. Unfortunately, the patient’s blood sample was not available for germline testing. However, the presence of a variant allele frequency (VAF) of nearly 50% in ETMR tumor of the patient 4 suggests that one of the DICER1 mutations is of germline origin. Specifically, the splicing mutation c.5365-2A>G has been reported to be a germline mutation [17], while D1709E c.5127T>A is reported to be a somatic mutation [18]. Therefore, the presence of second hit in patient 4’s DICER1 gene supports the involvement of DICER1 syndrome.

In the context of DICER1 syndrome, patient 4 may not be considered an extremely unusual case. Although most DICER1 syndrome-related tumors occur in adolescents and young adults (AYA), documented cases have shown multinodular goiter in individuals as old as 40 years, embryonal rhabdomyosarcoma (ERMS) of the cervix in individuals as old as 45, and differentiated thyroid carcinoma in individuals as old as 40 [19]. Patient 4 presents as a cautionary example for neuropathologists, highlighting that while ETMR cases predominantly occur in young patients, particularly in infant, young adults can also develop ETMR if they harbor DICER1 mutations. Considering the poor prognosis associated with ETMR, we propose that in adult patients who show morphological features suggestive of ETMR and exhibit LIN28A positivity, contrary to the current recommendation in the WHO CNS tumor classifications, germline testing for DICER1 mutation should be conducted concurrently with C19MC alteration testing, without waiting for a negative result on the C19MC molecular study. This expedited diagnosis would enable older patients to receive standard radiation therapy, which is typically not feasible for pediatric ETMR patient due to potential developmental impairment. Moreover, given that adult ETMR patients are of reproductive age, genetic counseling becomes particularly relevant in their care.

In conclusion, we have presented three cases of ETMR characterized by typical C19MC amplification, along with a DICER1-mutated ETMR, which exhibited distinct morphological features including chondroid differentiation. The case of the DICER1-mutated ETMR in a 30-year-old patient challenges both the diagnostic approach and the underlying biology of ETMR formation n adult individual. Given the absence of effective molecular studies of C19MC amplification and DICER1 mutations in adult CNS embryonal tumors that exhibit positive staining for LIN28A. This approach may provide valuable insight and potentially guide treatment strategies in these cases.

This study was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (Grant number: HI14C1277) and the Institute for Information & Communications Technology Promotion (IITP) grant funded by the Korean government (MSIP) (No.2019-0567).

S.-H.P. designed and supervised the study and E.E.K. wrote the entire manuscript. K.L. analyzed the methylation data and wrote the manuscript. S.-H.P. and E.E.K. reviewed histopathology slides and anonymized molecular data for qualitative analysis. J.-H. P. and M.-S.K. operated and treated the patients, and provided clinical data. H.Y. analyzed the NGS data derived from the NGS studies using a customized brain tumor targeted gene panel. All authors have reviewed and edited the final manuscript. All materials had been obtained for the electronic medical record of the patients, which were anonymized and retrospectively reviewed. No extra-human materials were obtained from the patients for this study. Under the Korean Bioethics and Safety Act, additional consent to publish was waived.

The authors declare no competing interests.

Clinicopathological features of three patients with TTYH1::C19MC fusion and one patient with DICER1 mutation