Embryonal tumor with multilayered rosettes: Post‐treatment maturation and implications for future therapy

Abstract Background Embryonal tumor with multilayered rosettes (ETMR) is a deadly grade IV pediatric brain tumor. Despite an intensive multimodal treatment approach that includes surgical resection, high‐dose chemotherapy, and radiotherapy, the progression‐free survival at 5 years is less than 30%. Case We report a case of long‐term survival in a 5‐month old female with a large mass in the posterior fossa, diagnosed as ETMR, which subsequently underwent treatment‐induced maturation. Prior to chemotherapy, histopathology revealed an abundance of highly proliferative, undifferentiated cells and multilayered rosette structures. Conversely, post‐treatment histopathology revealed cell populations that differentiated into neuronal and ganglionic phenotypes. At 5‐year follow‐up, the patient remains progression‐free. Conclusion This finding contributes to the few reports to date of post‐treatment differentiation/maturation of ETMR cell populations, with an implication for less cytotoxic therapeutic interventions aimed at differentiation.


| INTRODUCTION
Embryonal tumor with multilayered rosettes (ETMR) is a deadly pediatric brain tumor that predominantly affects infants younger than 3 years old and has a 5-year survival rate between 0% and 30%. 1,2 ETMR has low intertumoral genetic heterogeneity, is characterized by a hallmark amplification of the C19MC miRNA cluster on Chromosome 19q13.42, and has high expression of the RNA-binding protein, LIN28A. 3,4 The histological signature of ETMR is multilayered rosettes, which are remnants of the undifferentiated neural tube, suggesting a prenatal oncogenic transformation that results in embryonal progenitor and progenitor-like cells. 5 Stalled developmental programs were recently described as the root cause of pediatric brain tumors, including ETMR. Jessa et al. 6 mapped bulk and single-cell transcriptomes of ETMR and other pediatric brain tumors, juxtaposing this data to reference datasets taken from human fetal brain and their developmental atlas derived from the analysis of cell types and differentiation pathways in the prenatal mouse and human brainstem. While the cell of origin for ETMR was postulated to derive from an oncogenic transformation in prenatal radial glia cells, the bulk tumor microenvironment mapped to a spectrum of neuronal lineage populations, including a progenitor-like phenotype, a migratory phenotype, and a more mature phenotype. 6 The progenitor-like phenotype expressed the TTYH1-C19MC fusion, whereas this promotor region was found to be silenced in malignant cells that expressed neuronal differentiation markers. 6 The authors of this study provided a foundational framework for targeting restricted developmental pathways to overcome the impaired differentiation observed within ETMR and other pediatric brain tumors. 6 Herein, we report a case of long-term survival (greater than 5 years) in a patient with ETMR, whose post-treatment histopathol- After ruling out infectious and metabolic differentials with diagnostic laboratory tests, an MRI of the brain was obtained and showed a 6.5 Â 6.5 Â 4.4 cm calcified, heterogeneously enhancing mass in the right posterior fossa ( Figure 1A). Subsequent to subtotal resection, the residual tumor size was 4.9 Following diagnosis, the patient underwent 4 cycles of induction chemotherapy, according to the DFCI-IRS-III regimen. 7 Following the fourth chemotherapy cycle, MRI showed a 72% decrease in the tumor size ( Figure 1C), and a gross total resection of the tumor was performed thereafter ( Figure 1D).

| Defibrotide treatment
An adverse effect of this patient's chemotherapeutic course was sinusoidal obstructive syndrome, which was rapidly responsive to treatment with defibrotide. Defibrotide is a polydisperse polydeoxyribonucleotide that has thrombolytic effects that are beneficial for the hypercoagulable sequelae of cancer states, and it also demonstrated anti-tumor activity when used as a single agent against multiple myleoma. 8 Recently, Dong et al. 9 has shown defibrotide to modulate brain metastasis through inhibition of "proliferation, migration, invasion, and promotion of lactate dehydrogenase release of brain metastatic tumor cells, and elevation of the levels of blood brain barrier tight junction proteins and metastasisrelated proteins" via activation of adenosine A2A receptors and downstream inhibition of the SDF-1/CXCR4 axis. Lastly, defibrotide was shown to have anti-angiogenic properties after inhibiting the formation of new blood vessels during in-vitro and in-vivo studies and was postulated for use as an adjuvant anti-cancer therapeutic. 10 We are uncertain how this drug impacted the disease course in the patient presented here; however, defibrotide is noteworthy to consider for further investigation in embryonal CNS tumors.

| Pre-and post-treatment pathology demonstrating maturation
Following the first resection of tumor tissue, and prior to chemotherapy, pathological examination of the collected specimen revealed a plethora of characteristic multilayered rosettes in a

| DISCUSSION
The case presented here is the fourth known case in the literature describing a patient with ETMR, whose post-treatment pathology showed differentiation of tumor cells along their programmed lineage; this is in comparison to the pre-treatment pathology of rapidly dividing undifferentiated cells. Importantly, to the best of our knowledge, this is the only report of an ETMR patient with post-treatment differentiation of tumor cells who has exceeded the 5-year survival mark following initial diagnosis.
Of the three post-treatment differentiation cases published to date for ETMR, the study by Antonelli et al. 11 is the only other case reporting long-term patient survival. At the time of their publication, the patient was alive and well 31 months from initial diagnosis. 11 The authors presented a case of a 21-month-old male diagnosed with ETMR predominantly in the left fronto-opercular region. 11 Histopathology post-chemotherapy revealed the presence of neurons and ganglion cells within abundant neuropil, similar to the pathology findings reported here. 11 Multilayered rosettes and immature embryonal cells were not detected, and amplification of the C19MC locus was only detected in about 10% of tumor nuclei, compared to 60% in the pre-treatment tumor specimen. 11 Interestingly, post-treatment MRI of the case presented herein and the case presented by Antonelli et al. 11 both demonstrated a significant reduction in tumor following therapy.
We agree with the findings by Antonelli et al. 11  (PNET), but confirmed postmortem to be ETMR. While the pontine mass was slightly reduced in size after 6 months from the initial diagnosis and treatment, there was widespread dissemination elsewhere, and the patient eventually succumbed to her disease course. 12 Upon postmortem brain biopsy, there were regions of primitive, proliferating, undifferentiated cell populations, but also less cellular regions with glial and neuronal maturation in the setting of a rich neuropil landscape. 12 Only the primitive regions had strong immunoreactivity for LIN28A and only rare GFAP/synaptophysin positivity; the differentiated regions had strong immunoreactivity with MAP2 and GFAP, which are mature neuronal and glial markers, respectively, and these areas were completely negative for LIN28A. 12 Furthermore, Lafay-Cousin et al. 13 13 Ganglion cell morphology and a low Ki-67 index were both present, as was also seen in the case that we report here. 13 Additionally, LIN28 was predominantly negative in these tumor samples. 13 Following the second recurrence, however, the pathology resembled the pre-treatment pathology, with undifferentiated tissue, rosettes, and high LIN28 positive immunoreactivity. 13 14 The authors also reported six other cases in the literature of patients with medulloblastoma who had post-treatment maturation of tumor specimens into neuronal/glial, neuronal, gangliocytoma, ganglioneurocytoma, and ganglioglioma phenotypes across studies. 14 Additionally, Bidgoli et al. 15 reported on a 7-week-old female diagnosed with an embryonal tumor defined as PNET/cerebral neuroblastoma. Post-chemotherapy, the resected tumor tissue was no longer WHO Grade IV, but a WHO Grade I ganglioglioma, inferring maturation and differentiation from the original tumor tissue biopsied pre-treatment. 15 As with all of the cases discussed thus far, there was impressive regression of tumor bulk. Additionally, the patient was alive and well over 3 years from initial diagnosis at the time of publication. 15 Driever et al. 16 reported a case of a 5-year-old male who was diagnosed on histopathology with an undifferentiated WHO Grade IV PNET with focal neuronal differentiation. Following chemoradiation, a local recurrence of the tumor showed loss of the initial small, round, blue cells and expression of glial fibrillary acid protein (GFAP), inferring differentiation along the astrocytic lineage. 16 At the time of this publication, the patient was in complete remission 31 months from initial diagnosis. 16 Lastly, Nozza et al. 17 reported an 8-month-old male diagnosed with the malignant embryonal tumor, pineoblastoma, which histopathologically appears as small, round, blue cells akin to a PNET's histopathology. The histopathology seen on pre-treatment biopsy specimens was consistent with a pineoblastoma; however, following treatment with inductive and high-dose chemotherapy, repeat biopsy specimens revealed a neuronal tumor composed of ganglion and neurocytic cells, classified as a low-grade neuronal tumor. 17 The patient was still in remission 9 months following the end of therapy at the time of publication. 17 Our case contributes to the relatively few pieces of literature that elucidate the clinical association of post-treatment differentiation of tumor tissue on pathology, reduced tumor burden on imaging, and prolonged survival of the patient. A pre-clinical correlate can be seen with the findings of Jessa et al., 6 who found a common theme of stunted differentiation of neural progenitors across various pediatric brain tumors; when a knockout mutant tumor line was allowed to differentiate, there was a phenotypic switch towards mature and differentiated cells. Given such findings, these investigators suggest that future therapeutic development be aimed at the differentiation of tumor tissue. 6 Recently, brain-penetrating imipridones (ONC201 and ONC206) were studied in in vitro and in vivo models of diffuse midline glioma (DMG), and Przystal et al. 18 found that in a population of DMG cells that were treated with ONC201, there was a shift from an immature, undifferentiated oligodendrocyte-like progenitor cell phenotype to an increased population of differentiated cells along the astrocytic lineage. Importantly, the findings of Przystal et al. 18 led to the development of two clinical trials covering a number of different pediatric brain tumors.
In summary, we have presented a case with prolonged survival (>5 years) after post-treatment differentiation of the patient's pathology and reviewed similar accounts reported in the literature. These collective findings support the notion for the development of novel therapeutics that are less cytotoxic than chemotherapy, but that achieve the objective of inducing maturation of embryonal tumor tissue in order to potentiate cancer remission.

AUTHOR CONTRIBUTIONS
Francesca M. Gualano contributed to conceptualization, validation, writing (original draft, review, and editing), and visualization. Patrice Hassoun contributed to visualization and review. Claire L. Carter contributed to conceptualization, validation, writing (review and editing), visualization, supervision, and project administration. Derek Hanson contributed to conceptualization, validation, writing (review and editing), visualization, supervision, and project administration.