Extra-neural metastases in pediatric diffuse midline gliomas, H3 K27-altered: presentation of two cases and literature review

Introduction Pediatric diffuse midline gliomas (DMG), H3 K27- altered, are the most aggressive pediatric central nervous system (CNS) malignancies. Disease outcome is dismal with a median survival of less than one year. Extra-neural metastases are an unusual occurrence in DMG and have been rarely described. Methods and results Here, we report on two pediatric patients affected by DMG with extra-neural dissemination. Their clinical, imaging, and molecular characteristics are reported here. An 11-year-old male 5 months after the diagnosis of diffuse intrinsic pontine glioma (DIPG) developed metastatic osseous lesions confirmed with computed tomography (CT) guided biopsy of the left iliac bone. The patient died one month after the evidence of metastatic progression. Another 11-year-old female was diagnosed with a cerebellar H3K27- altered DMG. After six months, she developed diffuse sclerotic osseous lesions. A CT-guided biopsy of the right iliac bone was non-diagnostic. She further developed multifocal chest and abdominal lymphadenopathy and pleural effusions. Droplet digital polymerase chain reaction (ddPCR) on pleural effusion revealed the presence of H3.3A mutation (c.83A>T, p.K28M). The patient died 24 months after the diagnosis of DMG and 3 months after the evidence of metastatic pleural effusion. Discussion Extra-neural metastasis of DMG is a rare event and no standard therapy exists. An accurate and early diagnosis is necessary in order to develop a personalized plan of treatment. Further research is needed to gain further insights into the molecular pathology of DMG, H3K27- altered and improve the quality of life and the final outcome of patients with this deadly disease.


. Introduction
Pediatric diffuse midline gliomas (DMGs), H3 K27-mutant, are a rare group of malignancies, first introduced in the 2016 World Health Organization (WHO) Classification of Tumors of the Central Nervous System (CNS) with loss of H3p. K28me3 (K27me3) and usually an H3 c.83A>T p.K28M (K27M) substitution in one of the histone H3 isoforms (CNS WHO grade 4). In the 2021 WHO Classification of Tumors of the CNS, the DMGs have been renamed to "diffuse midline glioma, H3 K27-altered" to include additional molecular changes (such as aberrant overexpression of EZHIP, or an EGFR mutation) that also result in H3 K27 alterations (Louis et al., 2016(Louis et al., , 2021. The preferential location is the brainstem or the pons [the latter named diffuse intrinsic pontine glioma (DIPG)], or bithalamic, whereas DMGs in adolescents and adults predominantly arise unilaterally in the thalamus or in the spinal cord (Louis et al., 2021). On magnetic resonance imaging (MRI), DIPGs classically have their epicenter in the pons and typically involve >50% of its surface, often asymmetrically, with frequent encasement of the basilar artery (Steffen-Smith et al., 2014). There may be an exophytic component and/or infiltration into the midbrain, the cerebellar peduncles, and the cerebellar hemispheres. Thalamic tumors may be unilateral or bilateral, the latter being more frequent in the EGFR-mutant subtype (Broniscer et al., 2018). Although epidemiological data remain scant for DMG, the incidence of DIPG is estimated to be 0.54 cases per 1 million person-years overall and 2.32 cases per 1 million person-years in people aged ≤20 years, with no sex predilection (Mackay et al., 2017). DIPG represents 10%−15% of all pediatric brain tumors and 75% of all pediatric brainstem tumors. Thalamic DMGs are rarer, representing 1%−5% of pediatric brain tumors (25% of thalamic tumors) (Ryall et al., 2016). To date, there is no known specific genetic susceptibility for DMG, but exceptionally, DMGs may occur in the setting of a cancer predisposition syndrome such as Li-Fraumeni syndrome or mismatch repair deficiency. Independently from the location, the prognosis of DMG is poor, with a 2-year survival rate of <10% (Mackay et al., 2017). Large autopsy-based studies of DIPG have described leptomeningeal metastasis in 40% of cases, as well as diffuse spread to involve the thalamus, the cervical cord, and even the frontal lobe (Buczkowicz et al., 2014). To date, extra-neural metastases in patients with DMG, H3 K27altered, have been reported in 12 cases (Megan et al., 2018;Stephens et al., 2019;Bhatt et al., 2020;Handis et al., 2021;Li et al., 2021;Mohiuddin et al., 2021;Al Sharie et al., 2022;Lazow et al., 2022;Silva et al., 2022;Aftahy et al., 2023). In this study, we report two cases of pediatric DMG with extra-neural metastasis carrying H3.3K27 mutation: one patient was found to have osseous and bone marrow metastases and the second one showed multiple bone lesions, multifocal chest and abdominal lymphadenopathy, and metastatic pleural effusion.
. Case reports . . Case An 11-year-old boy was presented in June 2020 with a 1week history of diplopia due to VI cranial nerve (CN) deficit, headache, and asthenia. Brain computed tomography (CT) scan revealed an enlarged pons characterized by a diffuse hypodense alteration ( Figure 1A). Subsequently, MRI of the brain and the spine was performed. The MRI demonstrated the presence of an infiltrative mass involving more than 50% of the pons. MRI features were characteristic and consistent with the diagnosis of DIPG ( Figures 1B-F). The MRI excluded other brain and spine lesions referred to metastasis. The patient underwent a stereotactic biopsy of the lesion in accordance with the institutional protocol without complications. Histological examination confirmed the radiological diagnosis of DIPG, showing an infiltrative glial cell proliferation, with tumor cells displaying loss of H3K27me3 and expression of H3K27M-altered protein ( Figures 1G-I). In line with the immunohistochemical results, molecular analysis (polymerase chain reaction [PCR] and direct sequencing) documented the presence of H3F3A mutation (c.83A>T, p.K28M); neither activin receptor 1 (ACVR1) nor B-raf proto-oncogene (BRAF) mutations were identified. The patient started a 12-week induction regimen with vinorelbine and nimotuzumab, followed by local radiation therapy (volumetric modulated arc therapy [VMAT]) with 54 Gy in 1.8 Gy per fraction from the third week (Massimino et al., 2014;Massimino, 2022). Five months after the diagnosis of DIPG, the patient presented low back pain, bilateral lower extremity weakness and headache, suggestive of clinical progression. Brain and whole spine MRI demonstrated extensive leptomeningeal enhancement throughout the brain and spinal cord. Enhancing lesions throughout the vertebrae were also noted (Figures 2A-C). Because of the extent of the disease, whole-body CT was performed and revealed numerous osteoblastic lesions involving the vertebrae, sternum, and pelvis. A left iliac bone CT-guided biopsy was performed revealing bone metastases of DIPG ( Figures 3A-C). The immunohistochemical evaluation of the malignant cells revealed an expression for GFAP and H3K27M in association with H3K27me3 loss ( Figures 3D, E). The clinical course of the disease was rapidly progressive and fatal. The patient died 1 month after the evidence of the metastatic progression.

. . Case
An 11-year-old girl with no relevant family history was presented to our emergency room in March 2020 with a 1month history of vertiginous syndrome and sporadic vomiting. On examination, horizontal nystagmus and ataxia were documented. An urgent non-enhanced brain CT revealed a large and heterogeneous hypodense mass located within the fourth ventricle, which was slightly dilated (Figure 4a). She was therefore admitted to our hospital. Subsequently, a contrast-enhanced MRI of the brain and whole spine was performed. The MRI confirmed the presence of an infiltrative mass located in the fourth ventricle extending into the left lateral recess (Figures 4b-f). There were no other brain and spine lesions referred to metastases. After multidisciplinary discussion, neuronavigation and occipital craniotomy with tumor resection with direct cortical and subcortical stimulation were performed under general anesthesia. Compared to the first MRI study, MRI scanning within 24 h after surgery documented total resection. Microscopy on tissue sections showed a heterogeneous malignant neoplasm with palisading necrosis and extensive perivascular proliferation. Tumor cells ranged in size from small to medium size, with irregular hyperchromic nuclei and eosinophilic, scarce, or clear cytoplasm arranged in sheets and at the perivascular site ( Figure 4g). On . /fnmol. .

FIGURE
Computed tomography (CT) and pathology of iliac bone lesion of case . A bone biopsy was performed using an gauge coaxial bone needle (A, B). The lesion was so sti that the first needle bent, and a second approach was needed. The bone histology confirmed the skeletal metastases of DIPG (C), showing the presence of a bland spindle cell proliferation infiltrating the lamellar bone with positive IHC staining for GFAP (D) and H K M-altered protein (E).

. Discussion
Metastases of pediatric DMG outside the CNS are extremely rare. Since "diffuse midline glioma, H3K27M-mutant" was introduced as a distinct entity in the 2016 edition of the WHO classification of tumors of CNS, a total of 12 cases (3 M) aged 4-36 years of extra-neural metastases have been reported in the literature (Table 1). We presented an 11-year-old boy with DIPG  and an 11-year-old girl with cerebellum DMG who developed extra-neural metastases a few months after diagnosis, despite cancer treatment consisting of radiotherapy and concomitant nimotuzumab and vinorelbine (Massimino et al., 2014;Massimino, 2022). Localization of DMG in midline sites such pineal region, hypothalamus, and cerebellum is exceptional (Solomon et al., 2016;Meyronet et al., 2017;Nakata et al., 2017). In a series of 47 diffuse midline gliomas with histone H3K27M mutation, only one 9-year-old boy presented with a tumor arising in the cerebellum (Solomon et al., 2016). Similarly, in a retrospective series of 164 cases with molecularly confirmed H3K27M-mt DMGs, the cerebellum location was identified in only two patients (Zheng et al., 2022). Interestingly, Wang et al. (2018) showed that patients with H3K27M-mutant gliomas in unusual anatomical locations (cerebellum, corpus callosum, lateral ventricle, frontal, and temporal lobe) had a better prognosis compared with those with corresponding tumors in the brainstem. Recently, Hazaymeh et al. (2022) showed that patients affected by glioblastoma multiforme (GBM) undergoing gross total resection exhibited a significant survival benefit compared to their counterparts without gross total resection. Our cases 1 and 2 developed osseous metastases 5 months and 6 months, respectively, since the initial diagnosis. Osseous and bone marrow metastases of our case 1 were documented by CT-guided biopsy of the left iliac bone, and the patient died 1 month later. It is interesting to note that bone metastases are common sites of extra-neural involvement in high-grade gliomas (HGGs), such as DMG and GBM. This predilection of bone may come from both tumorderived and extracellular niche-derived cues. For example, many hematopoietic stem cell proteins are expressed by GBM cells, including stromal cell-derived factor 1 alpha (SDF-1α), C-X-C chemokine receptor type 4 (CXCR4), osteopontin (OPN), and cathepsin K (CATK) (Hira et al., 2018). Glioblastoma cells are also able to recruit bone marrow-derived progenitor cells providing a perivascular support role regulated by vascular endothelial growth factor (VEGF) (Burrell et al., 2014). In addition, CXCR4, OPN, CATK, and CD44 are induced by hypoxia-inducible factor-1α and VEGF, two proteins known to increase glioma aggressiveness and invasion (Colwell et al., 2017). In addition to bone metastases, our case 2 developed lymph nodes and pleura involvement. The diagnosis of multiple extra-neural metastases was more difficult since bone and lymph node biopsies and pleural fluid analysis were negative for cancer cells. The diagnosis was concluded due to positive ddPCR for H3.3A mutation on pleural effusion. Recently, Wolter et al. (2022) reported on the application and validation of a set of molecular assays for glioma diagnostics based on ddPCR, enabling the detection of diagnostically relevant gliomaassociated mutations in the isocitrate dehydrogenase (IDH)1, IDH2, H3-3A, BRAF, and protein kinase C alpha (PRKCA) genes, as well as in the telomerase (TERT) promoter and other relevant copy number alterations. Recently, Massimino et al. identified and validated a prognostic marker based on the expression of 13 circulating microRNAs in serum that can shed light on a patient's risk of progression (Iannó et al., 2022). Due to its rarity, the biological mechanisms behind tumor dissemination outside the CNS of DMG have not been welldescribed. Although improved care of patients affected by DMG is going to lead in some cases to longer survival, extra-neural metastases in DMG were detected at diagnosis or relatively early after diagnosis. Certainly, the absence of routine surveillance for    (Mistry et al., 2019). Increasing evidence from clinical and experimental studies suggests that surgical trauma caused by biopsies or resections may potentially lead to tumor progression and metastatic disease (Alieva et al., 2018). On hospital admission, both our patients underwent neurosurgical procedures: case 1 a stereotactic biopsy of the pontine lesion and case 2 a complete surgical resection of cerebellum mass. Recently, Massimino et al. showed that pediatric patients affected with pediatric DMG undergoing biopsies had more dissemination (P = 0.04) and less local progression (Massimino et al., 2014). Alieva et al. (2018) showed, in a retrospective analysis of GBM patients, an increase in tumor volume after biopsy. In mice, the cellular mechanisms mediating this response are dependent on inflammation, especially on the CCL-2-dependent recruitment of macrophages, which can be blocked by treatment with dexamethasone. The immune system may also have a role in tumor extra-neural dissemination. In patients affected by GBM, multiple hypotheses have been postulated as why, such aggressive tumors, only rarely exhibit metastases outside the brain. The main pathophysiological ideas are the "seed vs. soil" hypothesis (Mohme et al., 2017) that describes the preference of metastatic tumor cells to grow inside the brain where the local microenvironment is favorable, and the "peripheral immunosurveillance" hypothesis, which holds that the activated peripheral immune system is able to eliminate GBM tumor cells that left the immune protected brain microenvironment. However, the discovery of circulating tumor cells (CTCs) in up to 20% of GBM patients has renewed interest in this discussion. However, it remains unclear why the CTCs do not form extraneural metastases at the expected frequency. The occurrence of extracranial GBM metastases in recipients of organ transplantation from donors diagnosed with GBM points to a decisive role of the immune system in containing extracranial growth (Jimsheleishvili et al., 2014;Nauen and Li, 2014). Recently, Mohme et al. (2020) described a case of extracranial metastases from GBM during immunological remission of the intracerebral tumor with checkpoint inhibition. They postulated that the combination of functional impairment of the peripheral immune system, as reflected by a steady increase of exhaustion markers and the occurrence of metastasis with an increased mutational burden, enabled the extracranial dissemination and disease progression, while intracranial GBM could be controlled by checkpoint inhibition (Mohme et al., 2020). Similarly, our case 2 showed disease progression at bone sites and partial response of intracranial DMG to second-line treatment with craniospinal irradiation and systemic chemotherapy with irinotecan and bevacizumab regimen. At the time, neither an international nor European consensus chemotherapy regimen was universally agreed upon for DMG treatment, and the Phase 2 Children's Oncology Group (COG) study ACNS0126 demonstrated that single-agent TMZ during and after radiotherapy failed to improve pediatric HGG survival compared to historical controls (Jakacki et al., 2016). In Italy, the standard treatment of pediatric H3K27M-a DMG includes radiotherapy, concomitant nimotuzumab and vinorelbine, and reirradiation at relapse (Massimino, 2022). Recent studies support the bevacizumab regimen showing superior survival compared to historical-agent regimens and nearly all other published treatment strategies (Hummel et al., 2016;Lu et al., 2019). However, further research is needed to prove the safety and efficacy of bevacizumab in children and adolescents affected by DMG, H3 K27-altered.

. Limitations
Our study have some limitations, primarily due to the paucity of extra-neural DMG metastases in the literature. All the studies included in this review are case reports or case series. None of our patients completed genetic testing for germline mutations; therefore, familial predisposition cancer syndrome cannot be excluded.

. Conclusion
Extra-neural metastasis of DMG is a rare event. The present cases emphasize the need to consider unusual localization of pediatric brain cancers, especially because early diagnosis and active treatment may be crucial to improve prognosis and survival. Even if no standard therapy exists to treat extra-neural metastasis of DMG, it is mandatory to establish promptly an accurate and specific diagnosis in order to develop a personalized plan of treatment. Moreover, the biopsy of the primary and metastatic sites should be considered for any pediatric patients with DMGs due to the important prognostic implications as well as to develop more effective treatment strategies. Considering the rarity of extra-neural metastasis of DMGs, international registries and collaborative multicenter studies are warranted to gain further insights into the molecular pathology of DMG, H3K27-altered and improve the quality of life and the final outcome of patients with this deadly disease.

Data availability statement
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.