Clinically relevant molecular hallmarks of PFA ependymomas display intratumoral heterogeneity and correlate with tumor morphology.

Posterior fossa type A (PF-EPN-A, PFA) ependymoma are aggressive tumors that mainly affect children and have a poor prognosis. Histopathology shows significant intratumoral heterogeneity, ranging from loose tissue to often sharply demarcated, extremely cell-dense tumor areas. To determine molecular differences in morphologically different areas and to understand their clinical significance, we analyzed 113 PF-EPN-A samples, including 40 corresponding relapse samples. Cell-dense areas ranged from 0 to 100% of the tumor area and displayed a higher proportion of proliferating tumor cells (p < 0.01). Clinically, cell density was associated with poor progression-free and overall survival (pPFS = 0.0026, pOS < 0.01). Molecularly, tumor areas with low and high cell density showed diverging DNA methylation profiles regarding their similarity to distinct previously discovered PF-EPN-A subtypes in 9/21 cases. Prognostically relevant chromosomal changes at 1q and 6q showed spatial heterogeneity within single tumors and were significantly enriched in cell-dense tumor areas as shown by single-cell RNA (scRNA)-sequencing as well as copy number profiling and fluorescence in situ hybridization (FISH) analyses of different tumor areas. Finally, spatial transcriptomics revealed cell-dense areas of different tumors to be more similar than various different areas of the same tumor. High-density areas distinctly overexpressed genes encoding histone proteins, WNT5A, TGFB1, or IGF2. Relapsing tumors displayed a higher proportion of cell-dense areas (p = 0.036), a change in PF-EPN-A methylation subtypes (13/32 patients), and novel chromosome 1q gains and 6q losses (12/32 cases) compared to corresponding primary tumors. Our data suggest that PF-EPN-A ependymomas habor a previously unrecognized intratumoral heterogeneity with clinical implications, which has to be accounted for when selecting diagnostic material, inter alia, by histological evaluation of the proportion of cell-dense areas.

Recurrent atypical teratoid/rhabdoid tumors (AT/RT) reveal discrete features of progression on histology, epigenetics, copy number profiling, and transcriptomics.

Atypical teratoid/rhabdoid tumors (AT/RT) are the most common malignant brain tumors manifesting in infancy. They split into four molecular types. The major three (AT/RT-SHH, AT/RT-TYR, and AT/RT-MYC) all carry mutations in SMARCB1, the fourth quantitatively smaller type is characterized by SMARCA4 mutations (AT/RT-SMARCA4). Molecular characteristics of disease recurrence or metastatic spread, which go along with a particularly dismal outcome, are currently unclear. Here, we investigated tumor tissue from 26 patients affected by AT/RT to identify signatures of recurrences in comparison with matched primary tumor samples. Microscopically, AT/RT recurrences demonstrated a loss of architecture and significantly enhanced mitotic activity as compared to their related primary tumors. Based on DNA methylation profiling, primary tumor and related recurrence were grossly similar, but three out of 26 tumors belonged to a different molecular type or subtype after second surgery compared to related primary lesions. Copy number variations (CNVs) differed in six cases, showing novel gains on chromosome 1q or losses of chromosome 10 in recurrences as the most frequent alterations. To consolidate these observations, our cohort was combined with a data set of unmatched primary and recurrent AT/RT, which demonstrated chromosome 1q gain and 10 loss in 18% (n = 7) and 11% (n = 4) of the recurrences (n = 38) as compared to 7% (n = 3) and 0% (n = 0) in the primary tumors (n = 44), respectively. Similar to the observations made by DNA methylation profiling, RNA sequencing of our cohort revealed AT/RT primary tumors and matched recurrences clustering closely together. However, a number of genes showed significantly altered expression in AT/RT-SHH recurrences. Many of them are known tumor driving growth factors, involved in embryonal development and tumorigenesis, or are cell-cycle-associated. Overall, our work identifies subtle molecular changes that occur in the course of the disease and that may help define novel therapeutic targets for AT/RT recurrences.

Atypical neurofibromas reveal distinct epigenetic features with proximity to benign peripheral nerve sheath tumor entities.

BACKGROUND: Plexiform neurofibromas can transform into atypical neurofibromas (ANF) and then further progress to aggressive malignant peripheral nerve sheath tumors (MPNST). ANF have been described to harbor distinct histological features and frequent loss of CDKN2A/B. However, histological evaluation may be rater-dependent, and detailed knowledge about the molecular mechanisms of malignant transformation is scarce. In general, malignant transformation can be accompanied by significant epigenetic changes, and global DNA methylation profiling is able to differentiate relevant tumor subgroups. Therefore, epigenetic profiling might provide a valuable tool to distinguish and characterize ANF with differing extent of histopathological atypia from neurofibromas and MPNST. METHODS: We investigated 40 tumors histologically diagnosed as ANF and compared their global methylation profile to other peripheral nerve sheath tumors. RESULTS: Unsupervised class discovery and t-SNE analysis indicated that 36/40 ANF cluster with benign peripheral nerve sheath tumors with clear separation from MPNST. 21 ANF formed a molecularly distinct cluster in proximity to schwannomas. Tumors in this cluster had a frequent heterozygous or homozygous loss of CDKN2A/B and significantly more lymphocyte infiltration than MPNST, schwannomas, and NF. Few ANF clustered closely with neurofibromas, schwannomas, or MPNST, raising the question, whether diagnosis based on histological features alone might pose a risk to both over- and underestimate the aggressiveness of these lesions. CONCLUSIONS: Our data suggest that ANF with varying histological morphology show distinct epigenetic similarities and cluster in proximity to benign peripheral nerve sheath tumor entities. Future investigations should pay special respect to correlating this methylation pattern to clinical outcomes.