Infant medulloblastoma (iMBL) is predominantly desmoplastic nodular (DN) or medulloblastoma with extensive nodularity (MBEN) histology, sonic hedgehog (SHH) driven. Intensified chemotherapy (intraventricular chemotherapy or high-dose chemotherapy with autologous stem cell rescue) is recommended to compensate for the omission of radiation. This study aims to analyze the outcomes of iMBL treated without treatment intensification. This retrospective study was performed on 28 patients with iMBL (children with medulloblastoma <3 years at diagnosis or 3-4 years with DN/MBEN histology) treated between January 2004 and April 2024 in a tertiary care institute in southern India. The median age at diagnosis and duration of symptoms were 24.5 months and 1 month, respectively. Vomiting (82%) and ataxia (71.4%) were the common presentations. Ventriculoperitoneal shunt or endoscopic third ventriculostomy was done in 57% of patients. Twenty-four patients (∼86%) underwent gross/near-total resection. The most common surgical complication was cerebellar mutism (n = 6, 21%). Half of the cohort had nonmetastatic disease. Sixteen patients had DN/MBEN histology. Only 68% (n = 19) opted to continue postoperative treatment. Chemotherapy protocols used were baby SFOP, HIT SKK, and modified Packer. One fourth (n = 7) received radiation (DN/MBEN = 3; other histology = 4) as a part of their initial treatment. There was no treatment-related mortality. The 5-year event-free survival (EFS) of patients who received postoperative chemotherapy ± RT was 70% (those with DN/MBEN were 82.5% and non-DN/MBEN were 35.7%). The 5-year EFS of patients with DN/MBEN histology who received only chemotherapy (n = 9) was 88.9%. Managing medulloblastoma in infants is challenging in LMIC, as a significant proportion of families opt against postoperative treatment. Although traditionally considered high risk, good survival can be achieved with non-intensified therapy without an increase in treatment-related mortality, especially in patients with DN/MBEN histology.

T cells and tumor-associated macrophages (TAMs) are critical immune components within the brain tumor microenvironment (TME), yet their precise roles in medulloblastoma remains unclear. In this study, we examined the infiltration characteristics of T cells in medulloblastoma tissues and analyzed the correlation between T cells and the clinical outcomes of medulloblastoma patients. Additionally, we further investigated the relationship between T cells and TAMs. We enrolled a total of 72 patients diagnosed with medulloblastoma and subsequently detected the T cell makers and programmed death 1/programmed death-ligand 1 (PD-1/PD-L1) in paraffin-embedded sections using multiple immunofluorescence staining method. The correlation between T cell infiltration, clinical characteristics and prognosis were analyzed. Finally, we used Spearman correlation analysis to evaluate the correlation between T cells and TAMs. The median age at diagnosis of 72 patients (54 boys, 18 girls) was 7.5 years (range: 0.8-18 years). These patients included 43 cases of classic medulloblastoma (CMB), 24 cases of desmoplastic/nodular medulloblastoma (DNMB), 2 cases of medulloblastoma with extensive nodularity (MBEN) and 3 cases of large-cell/anaplastic medulloblastoma (LCA). The molecular subgroups consisted of 3 wingless (WNT), 29 sonic hedgehog (SHH) and 40 non-WNT/non-SHH cases. Twenty-five cases presented with metastasis at diagnosis, while 47 cases were without metastasis. Thirteen cases exhibited with high-risk genetic abnormalities. The total T cells (P = 0.031) and CD4 T cells (P = 0.045) were significantly elevated in the SHH subgroup compared to those in the non-WNT/non-SHH subgroup. Patients with increased CD4 T cells had better 5-year PFS (P = 0.000) and OS (P = 0.001), while patients without metastasis showed better 5-year PFS (P = 0.031) and OS (P = 0.015). Multivariate analysis showed that CD4 T cells were an independent prognostic factor affecting both the 5-year PFS (P = 0.004, HR = 0.230, 95% CI = 0.085-0.662) and OS (P = 0.017, HR = 0.180, 95% CI = 0.044-0.739). Additionally, it was observed that CD4 T cells exhibited a positive correlation with Mtotal (total macrophages) (P < 0.05, r = 0.249) and Mmix (M1/M2 mixed phenotype macrophages) (P < 0.01, r = 0.325), and CD3+CD8+PD-1+ cells showed a positive correlation with Mmix (P < 0.05, r = 0.258). The increase in CD4 T cells predicts a better prognosis in medulloblastoma patients, particularly within the SHH and non-WNT/non-SHH subgroups, and they may serve as a potential therapeutic target for medulloblastoma. Additionally, there may be a potential interaction between CD4 T cells and TAMs that warrants further investigation.

Spatial transcriptomics technologies are revolutionizing our understanding of intra-tumor heterogeneity and the tumor microenvironment by revealing single-cell molecular profiles within their spatial tissue context. The rapid development of spatial transcriptomics methods, each with unique characteristics, makes it challenging to select the most suitable technology for specific research objectives. Here, we compare four imaging-based approaches-RNAscope HiPlex, Molecular Cartography, Merscope, and Xenium-alongside Visium, a sequencing-based method. These technologies were employed to study cryosections of medulloblastoma with extensive nodularity (MBEN), a tumor chosen for its distinct microanatomical features. Our analysis reveals that automated imaging-based spatial transcriptomics methods are well-suited to delineate the intricate MBEN microanatomy and capture cell-type-specific transcriptome profiles. We devise approaches to compare the sensitivity and specificity of different methods, along with their unique attributes, to guide method selection based on the research objective. Furthermore, we demonstrate how reimaging slides after the spatial transcriptomics analysis can significantly improve cell segmentation accuracy and integrate additional transcript and protein readouts, expanding the analytical possibilities and depth of insight. This study underscores important distinctions between spatial transcriptomics technologies and offers a framework for evaluating their performance. Our findings support informed decisions regarding methods and outline strategies to improve the resolution and scope of spatial transcriptomic analyses, ultimately advancing spatial transcriptomics applications in solid tumor research.

Tumor-associated macrophages (TAMs) constitute a significant proportion of the immune cell population within brain tumors. The polarization of macrophages exerts an important influence on the tumor microenvironment (TME). Nevertheless, the specific role of TAMs in sonic hedgehog (SHH) medulloblastoma remains unclear. To investigate the polarization characteristics and effects of TAMs in SHH medulloblastoma, we evaluated the infiltration of M1 and M2 macrophages in SHH medulloblastoma tissues and analyzed the correlation between TAMs recruitment and the clinical outcome of SHH medulloblastoma patients. We enrolled a total of 42 patients diagnosed with SHH medulloblastoma. Using multiple immunofluorescence staining on paraffin-embedded sections, we detected the activated phenotype (M1/M2) by monoclonal antibodies for CD68, HLA-DR and CD163. Subsequently, we analyzed the correlation between TAMs and clinical characteristics as well as prognostic factors. The median age of 42 patients (31 boys, 11 girls) was 5.3 years (range: 0.8-15.1 years). All patients had confirmed pathological types, including 4 cases of classic medulloblastoma (CMB), 33 cases of desmoplastic/nodular medulloblastoma (DNMB), 3 cases of medulloblastoma with extensive nodularity (MBEN), and 2 cases of large-cell/anaplastic medulloblastoma (LCA). Thirteen cases presented with metastasis at diagnosis, while twenty-nine cases were without metastasis. Four cases had high-risk genetic abnormalities. Different proportions of macrophages were found in the collected medulloblastoma tissues, and large amounts of CD68+HLA-DR+CD163+ cells were found. The study revealed that Mtotal (total macrophages) and Mmix (CD68+HLA-DR+CD163+ cells) were significantly higher in group of patients <5 years old (P < 0.05), and Mtotal in non-metastatic group were significantly higher than that in metastatic group (P = 0.043). M2 macrophages in CMB group were significantly higher than that in DNMB/MBEN group (P = 0.036), M1 macrophages were significantly higher in children without high-risk genetic abnormalities (P = 0.007). Five-year PFS was significantly poorer in patients ≥5 years old and metastatic group (P < 0.05). High Mtotal group had a better 5-year PFS (P = 0.000), whereas high M2 group had both better 5-year PFS and OS (P = 0.001, P = 0.001). Multivariate analysis showed that Mtotal and M2 macrophages were independent prognostic factors for 5-year PFS, and M2 macrophages were an independent prognostic factor for 5-year OS. The increase in total macrophages and M2 macrophages predicts a better outcome of SHH medulloblastoma. TAMs especially M2 macrophages might be a therapeutic target for SHH medulloblastoma.

Medulloblastomas with extensive nodularity are cerebellar tumors characterized by two distinct compartments and variable disease progression. The mechanisms governing the balance between proliferation and differentiation in MBEN remain poorly understood. Here, we employ a multi-modal single cell transcriptome analysis to dissect this process. In the internodular compartment, we identify proliferating cerebellar granular neuronal precursor-like malignant cells, along with stromal, vascular, and immune cells. In contrast, the nodular compartment comprises postmitotic, neuronally differentiated malignant cells. Both compartments are connected through an intermediate cell stage resembling actively migrating CGNPs. Notably, we also discover astrocytic-like malignant cells, found in proximity to migrating and differentiated cells at the transition zone between the two compartments. Our study sheds light on the spatial tissue organization and its link to the developmental trajectory, resulting in a more benign tumor phenotype. This integrative approach holds promise to explore intercompartmental interactions in other cancers with varying histology. In 2016, the World Health Organization (WHO) published a revised classification of central nervous system (CNS) tumors using molecular parameters. In this classification, some tumors previously recognized in the 2007 classification had been renamed or eliminated. The primitive neuroectodermal tumor (PNET) is no longer recognized. CNS embryonal tumors are now classified using specific genetic/molecular characteristics. Using molecular analysis, many tumors that were previously reported as PNET are now reclassified into know tumors with specific genetic characteristics.  Medulloblastomas are the most common embryonal tumors and have their own genetic/molecular defined groups (SHH-activated TP53-mutant, SHH-activated TP53-wildtype, WNT-activated, non-SHH/WNT group three, and non-SHH/WNT group four) in addition to their histologically defined groups (classic, desmoplastic/nodular, medulloblastoma with extensive nodularity, and large cell/anaplastic). A combination of the genetic profile and the histology determines the prognosis of these tumors. The sonic hedgehog protein, important in cell specialization and growth, is encoded in the SHH gene at chromosome 7. The WNT gene family in the chromosome 12q13 region encode for signaling proteins that regulate cell proliferation, cell fate, and embryogenesis. All other embryonal tumors are classified depending if they have an amplification of the C19MC region (19q13.42) on chromosome 19. If they have the amplification, they are called embryonal tumor with multilayered rosettes, C19MC-altered (WHO 9478/3*). If they do not have the amplification, they are called embryonal tumor with multilayered rosettes, NOS (WHO 9478/3). The other embryonal tumors are classified as medulloepithelioma (9501/3), CNS neuroblastoma (9500/3), CNS ganglioneuroblastoma (9490/3), and atypical teratoid/rhabdoid tumor (ATRT) with alterations of INI1 or in rare occasions BRG1. Immunohistochemical staining for INI1 or BRG1 expression is used to distinguish them. Those tumors that do not have alterations of either INI1 or BRG1 are classified as embryonal tumors with rhabdoid features (WHO 9508/3). Those embryonal tumors without amplification of the C19MC, no alterations of INI1 or BRG1, and no rosettes, are called embryonal tumor, NOS (WHO 9473/3). This last group is a wastebasket category for the tumors previously called PNET, which can not be classified under a genetic/molecular group. In tumors with the “NOS” designation (not otherwise specified), there is insufficient information for classification. Some of these tumors were previously named embryonal tumor with abundant neuropil and true rosettes. Concurrent with the publication of the WHO 2016 classification, a group recognized four new categories for PNET. They reviewed their previously classified PNET database and found that based on molecular analysis of the tumor samples, most of them were indistinguishable from known CNS supratentorial tumors and were classified into those known CNS supratentorial tumors. The small group of other tumors which can not be attributed to known CNS supratentorial tumors were classified into the four new categories. The new categories include CNS neuroblastoma with Forkhead Box R2 (FOXR2) activation, CNS Ewing sarcoma family tumor with CIC alteration, CNS high-grade neuroepithelial tumor with meningioma 1 gene (MN1) alteration, and CNS high-grade neuroepithelial tumor with BCL6 Corepressor (BCOR) alteration. The small number of tumors that did not show any molecular specificity to be included in a specific category were called CNS embryonal tumors, NOS. This group is similar to the wastebasket category of the WHO 2016 classification. The significance of these results have yet to be analyzed and incorporated in a future WHO classification. These four new groups had also been confirmed by a Polish study in 2020.